Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini. Opisthorchis viverrini and Clonorchis sinensis are pathologically important members of the family Opisthorchiidae. Chronic infections with these liver flukes closely associate with the development of the bile duct cancer (cholangiocarcinoma) and the liver cancer (hepatocarcinoma) in humans [ C. sinensis infection was 0.58% in 356,629 residents from 688 sampled pilot sites in China [ O. viverrini is considered an important food-borne parasitic disease in Southeast Asia including Thailand, Lao PDR, Vietnam, and Cambodia; approximately 67.3 million people are at risk of the infection [ O. viverrini and C. sinensis, which are classified by the International Agency for Research on Cancer as a group 1 biological carcinogen in 2009 [n humans . In a re in 2009 .C. sinensis and O. viverrini have similar life cycles, locations, pathogenicity and their morphologies, in particular the forms of their eggs and metacercariae differ only slightly [ C. sinensis and O. viverrini seem to differ; for example, cholelithiasis is one of the most serious complications of clonorchiasis but a rare complication of opisthorchiasis [ O. viverrini. From an epidemiological perspective, C. sinensis will make control more challenging.slightly . Howeverrchiasis . AlthougOpisthorchis/Clonorchis-like” eggs [A number of methods have been developed to detect these two species, such as direct microscopy, ELISA , conventke” eggs . Crude oke” eggs , 12. Botke” eggs . C. sinensis and O. viverrini.Recently, high resolution melting (HRM) for fast, high-throughput analysis of many pathogens has been developed, for example, variation scanning , species C. sinensis were collected from naturally infected cats in Guangdong Province. Adult worms of O. viverrini were kindly provided by Professor Sung-Jong Hong, Department of Medical Environmental Biology, Chung-Ang University College of Medicine, Republic of Korea, which were originated from the Lao PDR. Several closely related trematodes infecting humans were included as “heterologous” control samples for assessing the specificity of the real-time PCR assay, namely, Fasciola hepatica, Fasciola gigantica, Schistosoma mansoni, and Schistosoma japonicum. All parasite materials were preserved in 70% ethanol and kept at −20°C until the extraction of genomic DNA. Metacercariae of C. sinensis were collected from the fish muscles essentially according to a previously reported method [Adult worms ofd method . Fecal sμL of a lysis solution (ATL buffer-Qiagen); then 20 μL proteinase-K (50 g/mL) was added and incubated at 56°C for 2-3 h with brief vortexing every 30 min. For eggs, the incubation time was set 1-2 h longer. The mixture, after adding 200 μL buffer AL (Qiagen) containing guanidine hydrochloride and 4 μL RNase A (100 mg/mL) and mixing by pulse-vortexing for 15 s, was further incubated at 70°C for 10 min. Thereafter, 200 μL of ethanol (96–100%) was added and mixed by vortexing for 15–20 s. The contents were then loaded onto a QIAamp Spin Column for DNA binding and spin down for 1 min. The column with the DNA bound was washed several times using solutions  provided according to manufacturer's instruction. Finally, the genomic DNA was eluted in 50 μL elution buffer (AE) and stored at −20°C until use. The DNA from fecal samples was extracted using QIAamp DNA Stool Mini Kit according to the manufacturer's instructions, and the DNA from metacercariae in fish tissue was extracted according to a previous description [Total genomic DNA was extracted from adult worms, using the commercial QiAamp DNA extraction kit (Qiagen). In the case of adult worms, only a single specimen was used in each DNA extraction. Briefly, the sample was enzymatically digested in 180  ethanol 6–100% wacription . The con C. sinensis and O. viverrini (COX1e-F: 5′-GGTAGGGTGGTTTGAGC-3′ and COX1e-R: 5′-TCATAGTAACCGAGCTAAA-3′) was synthesized by Takara Biotechnology . Real-time PCR amplification was performed in Lightcycler 480 . The total reaction volume was 20 μL, which consisted of 1x Fast-Plus EvaGreen qPCR Master Mix , reaction buffer containing dNTPs, MgCl2, fast-activating chemically modified hot start enzyme, Cheetah Taq, 0.3 μM of each primer, and 1 μL genomic DNA as template.A pair of primers targeting the COX1 gene ofdF/dT). The HRM profile was then analyzed using HRM analysis software, as reported previously [The PCR was carried out with initiation at 95°C for 60 s, then 45 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 10 s, and extension at 72°C for 20 s. When PCR amplification was completed, HRM analysis was performed by lowering the temperature to 60°C for 5 min, followed by increasing the temperature ramping from 60° to 95° at 0.11°C/s, 25 acquisitions/°C. In this process, the PCR amplicons were allowed to denature and reanneal in fluorescence with changes in temperature  is y = −1.8603 log(x) + 22.815(R2 = 0.9978), when tested with C. sinensis (y = −1.5718 log(x) + 22.203(R2 = 0.9938), when tested with O. viverrini  and quantified by spectrophotometry. The 10-fold diluted DNA was performed and good result acquired. The formula of Cp value and template concentration  for fish samples, which was consistent with that of a previous study [It seems that the sensitivity ofus study .Tm) values and normalized melting curves. Constant HRM profiles with distinct Tm peaks were persistently obtained for all positive samples. As shown in  C. sinensis samples had a Tm of 78.05 ± 0.07°C, while the amplification product from 10 O. viverrini samples had an average 80.89 ± 0.06°C. As shown in  C. sinensis and the other represented O. viverrini. To ensure the accuracy of the method, the amplified products were sequenced in both directions. The results showed that all sequences were uniform with the HRM analysis result.The sequence difference between the forward and reverse primers will bring different melting temperature (μL to 1 pg/μL) of the template appeared to be reliable, while the profile is unsatisfactory when the template concentrations were ≤1 pg. The melting peaks were obtained from each dilution (10 ng/μL to 1 pg/μL), and it shows that Tm of C. sinensis was 78.97 ± 0.11, while Tm of O. viverrini was 80.92 ± 0.08°C  curve is as sensitive as 0.01°C, so single base can also be differentiated [The method established in the present study has an accuracy of 100% in reference to microscopic methods. Moreover, the detection limit of the method was below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria, which was equal to probed real-time PCR  and supeentiated .O. viverrini, C. sinensis, and Opisthorchis felineus are the three most medically important species in the family Opisthorchiidae [ O. felineus are absent. However, once the sequence difference between three species was proved enough, a multiplex real-time PCR for three liver flukes will be developed in the near future.rchiidae . Now, we O. viverrini and C. sinensis were not able to be differentiated because of high homology [A previous molecular method targeting the second internal transcribed spacer (ITS2) of rDNA showed good sensitivity, buthomology . Here, w C. sinensis and O. viverrini simultaneously. It offers a potential means for detection of these flukes in the intermediate hosts. Moreover, it would also help in determining the worm burden in fish or infected individuals in endemic areas and would therefore be a useful tool for epidemiological surveys.The present study established a method based on real-time PCR and HRM analysis for the rapid identification and differentiation of


Separation was achieved using an ACQUITY UPLC BEHC18 column  with a gradient elution program consisting of acetonitrile and 0.1% phosphoric acid solution. The detection wavelengths were set at 203, 254, 222, and 267 nm. The method was validated for linearity, accuracy, precision, limit of detection, and limit of quantification. The validated method was successfully applied to the simultaneous quantification of ten active compounds from several finished batches of SJZT. This validated that UPLC method is expected to provide a new basis for the quality control of SJZT.Sijunzi decoction (SJZT), a traditional Chinese formula (TCMF) consisting of four herbs, has been widely used for the treatment of various gastrointestinal symptoms. However, its modernization process is hindered by the lack of a powerful quality control method that covers the major active components in the formula. The aim of this study was to establish a UPLC method for the quantitative determination of ten active components in Sijunzi decoction including ginsenoside Rg Radix Ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae, and Radix Glycyrrhizae. In China, SJZT has long been used for the treatment of gastrointestinal disorders such as chronic gastritis and gastric and duodenal ulcer, and it could effectively attenuate nausea, vomiting, and diarrhea [Traditional Chinese herbal formulation (TCMF) has been widely used in the clinic for its well-proven efficacy with few side effects. Sijunzi decoction (SJZT) is one of the most famous TCMFs consisting of four herbs:diarrhea . Clinicadiarrhea . More rediarrhea . Moreovediarrhea .1, Re, Rf, Ro, Rb1, Rc, Rb2, and Rd) and glycyrrhizic acid were identified through structural elucidation [ Radix Ginseng [ Poria cocos [ Glycyrrhiza uralensis [Supporting these well-confirmed pharmacological efficacies, recent years have seen an increasing knowledge of the chemical components from SJZT. HPLC-MS was exploited to analyze the major components of SJZT, and eight ginsenosides . Specifically, ginsenoside Rgc effect . The pot Radix Ginseng, Poria cocos, Rhizoma Atractylodis Macrocephalae, and Glycyrrhizae uralensis, were purchased from Nanjing Traffic Hospital . All samples were identified by one of the authors (Professor Wang Xiao-Long) as authentic herbal medicine. Ginsenoside Rg1, Re, and Rb1 were purchased from Jilin University ; liquiritin, liquiritigenin, and glycyrrhizic acid were purchased from Chinese Food and Drug Inspection Institute; Atractylenolide III, Atractylenolide I, Atractylenolide II, and pachymic acid were purchased from Sichuan Weikeqi Biological Co., Ltd . The ten compounds used in the analysis were of analytical grade and their purity was more than 98%. Their chemical structures are shown in The four crude herbs,μm ACQUITY BEH C18 column with a flow rate of 0.3 mL min−1. Separation was achieved using a gradient method. Acetonitrile (A) and 0.1% phosphoric acid (B) were selected and the gradient solvent system was as follows: 0–5 min, (A) 19%; 5–13 min, 19%–30%; 13–25 min, 30%–50%; 25–33 min, 50%–60%; 33–37 min, (A) 60%–68%; 37–39 min, (A) 68%–19%; and 30–40 min, (A) 19%. The injection volume of samples was 5 μL and temperature of the column oven was maintained at 30°C. The UV wavelength was set at 203 nm , 254 nm , 222 nm (for atractylenolide I and III), 276 nm (for atractylenolide II), and 242 nm (for pachymic acid).The analytes were separated on an ACQUITY UPLC H-class system with a PDA detector using 2.1 mm × 100 mm × 1.7  Radix Ginseng (100 g), Poria cocos (100 g), Rhizoma Atractylodis Macrocephalae(100 g), and Glycyrrhizae uralensis (50 g) were crushed into small pieces and then mixed and decocted twice in 3500 mL water for 1 h in a glass flask. The decoction was filtered through 8 layers of gauze; the filtrate was concentrated in vacuum at 60°C at a final concentration of 2 g/mL.According to the original composition of SJZT, the four constituting herbs includingμL supernatant was injected into chromatographic systems for analysis.Ethanol was added to an aliquot of 5 mL SJZT overnight in order to remove the polysaccharides. The supernatant was transferred into a test tube and evaporated to dryness with vacuum at room temperature. Finally, the residue was reconstituted in 5 mL methanol by vortex mixing for 5 min and centrifuged at 16,000 rpm for 10 minutes. 5 1 (300.00 μg/mL), ginsenoside Re (302.10 μg/mL), ginsenoside Rb1 (300.00 μg/mL), liquiritin (90.30 μg/mL), liquiritigenin (87.00 μg/mL), glycyrrhizic acid (110.00 μg/mL), atractylenolide III (59.00 μg/mL), atractylenolide I (71.40 μg/mL), atractylenolide II (50.60 μg/mL), and pachymic acid (200.00 μg/mL) were prepared in methanol. These solutions were stored at 4°C and were stable for at least 1 month.The standard stock solutions of ginsenoside RgThe negative control samples of SJZT were prepared by deriving one herb from the prescriptions. The herbs were accurately weighed according to the prescription of SJZT and prepared with the same procedure as for the sample preparation.Specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision (repeatability and intra- and interassay), and accuracy (recovery) of this UPLC method were evaluated in accordance with International Conference on Harmonization (ICH) .1, ginsenoside Re, ginsenoside Rb1, liquiritin, glycyrrhizic acid, liquiritigenin, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid). The lower limit of quantification (LLOQ) was determined as the lowest concentration point of the standard curve and the signal-to-noise ratio was higher than 10. The lower limit of detection (LLOD) was defined as the amount that could be detected with a signal-to-noise ratio of 3.The standard calibration curve for the linearity assay was prepared with seven different concentrations of diluted standard solutions  were prepared. The quantity of each component was determined by the respective calibration curve. RSD was used to measure precision. The interbatch reproducibility test was carried out on three different batches.Recovery studies were carried out by spiking three concentrations of mixed standards at low (50% of the known amounts), medium (100% of the known amounts), and high (200% of the known amounts) in the 5 mL of SJZT. Then, the spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above.18 column  with acetonitrile and 0.1% phosphoric acid/water was selected as the preferred chromatographic conditions.Optimization of the separation conditions for HPLC analysis was performed including the mobile phase composition, gradient elution program, and wavelength. To obtain chromatograms with better resolution of adjacent peaks within shorter time, the chromatographic conditions were optimized. Methanol and acetonitrile were compared in the experiment. The result showed that acetonitrile was much better as it could result in a better resolution and shorter time for analysis. In addition, water and 0.1% phosphoric acid/water were investigated and the result showed that 0.1% phosphoric acid/water was better than water. As a result ACQUITY UPLC BEH CMoreover, different gradient profiles were also optimized. Actually, we tried to simplify the gradient elution system and shorten the analysis time, but peaks for atractylenolide III and atractylenolide I have not been completely separated except for the current condition to in the gradient program mentioned above.1, Re, and Rb1 (203 nm), liquiritin, glycyrrhizic acid, and liquiritigenin (254 nm), atractylenolide I and atractylenolide III (222 nm), atractylenolide II (276 nm), and pachymic acid (242 nm).In this experiment, the specificity of UV absorption was also investigated, using the present chromatographic conditions and comparing a SJZT sample with a standard mixture. The UV absorbance and the best UV detection wavelength of each compound in SJZT were confirmed as follows: ginsenoside RgThe validation study allowed the evaluation of the method for its suitability for routine analysis.Representative chromatograms of the standard solution, sample solution, and negative control samples at different UV wavelength were shown in 1, ginsenoside Re, ginsenoside Rb1, liquiritin, glycyrrhizic acid, liquiritigenin, atractylenolide I, atractylenolide II, atractylenolide III, and pachymic acid and the observed concentrated ranges were as follows: 4.68∼300.00, 4.72∼302.10, 4.68∼300.00, 1.41∼90.30, 1.72∼110.00, 1.36∼87.00, 1.12∼71.40, 0.79∼50.60, 0.92∼59.00, and 3.13∼200.00 μg/mL, respectively. The correlation coefficient (R2) of all biomarkers has good linearity of over > 0.9996 in the aforementioned ranges. The LODs and LOQs of the ten analytes were 0.01∼0.07 and 0.03∼0.23 μg/mL, respectively (The linearity of the developed method was assessed using seven different concentrations each of ginsenoside Rgectively .n = 6), the RSD of the SJZT repeatability, and stability test was in the range of 0.20–0.89% and 0.57%–1.04%, respectively  of intra- and interday was in the range of 0.21–1.18% and 0.20–1.10% (ectively . None ofThe accuracy of the method was assessed by a recovery assay. The spiked samples were then extracted, processed, and quantified in accordance with the methods mentioned above. The measured data showed that the recovery of the investigated components ranged from 95.07% to 102.67%, and their RSD values were all less than 3.0% . RecoverThe established analytical method was subsequently applied for the simultaneous determination of the ten markers in 3 batches of SJZT. The results are presented in In this study, a UPLC method for the simultaneous determination of ten active ingredients in SJZT has been developed and the results showed that it could be used for the quality control of the SJZT. Thus, this validated that UPLC method could be expected to provide a new basis for the quality control of SJZT.


To evaluate the role of intensity modulated radiotherapy (IMRT) for locally advanced pancreatic cancer (LAPC) and metastatic pancreatic cancer (MPC), and the prognostic factors in the setting of multidisciplinary approach strategies.63 patients with LAPC and MPC receiving IMRT in our institution were retrospectively identified. Information on patient baseline, treatment characteristics and overall survival (OS) time were collected. Data of pain relief and toxicity were evaluated. Univariate and multivariate analyses were conducted to investigate the prognostic factors.p = 0.029). Symptomatic improvements were observed in the 44 patients with abdominal/back pain after radiotherapy (RT) or concurrent chemoradiotherapy (CCRT), particularly in those with severe pain. Only 13.9% and 14.8% cases presented Grade ≥ 3 hematologic toxicities in RT and CCRT group, while no cases developed Grade ≥ 3 non-hematologic toxicities in both groups. Multivariate analysis indicated that tumors located in pancreas body/tail , pretreatment CA19-9 < 1000 U/mL  and concurrent chemotherapy  were independent favorable predictors for OS.All patients received IMRT with a median dose of 46.0 Gy. The median OS for LAPC and MPC patients were 15.7 months and 8.0 months, respectively (CCRT further improved OS for LAPC and MPC with acceptable toxicities, and use of RT markedly alleviated pain. Tumors located in pancreas body/tail, pretreatment CA19-9 level of < 1000 U/mL and CCRT were associated with better OS. However, regional intra-arterial chemotherapy did not show any survival benefit in our study. Pancreatic cancer is one of the deadliest human malignancies with a 5-year survival rate of less than 5% . AlthougAt the time of diagnosis, about 30% of patients present as locally advanced pancreatic cancer (LAPC), with a poor median survival of 10–12 months . HistoriThe objective of this study is to evaluate the efficacy of RT and the prognostic factors in the setting of multidisciplinary approach strategies and to assess the effect of RT on pain relief in patients with LAPC and MPC.This is a retrospective patient series and the patient selection criteria were: (1). Histologically or cytologically confirmed, or clinically diagnosed according to our clinical diagnosis criteria, which included typical pancreatic cancer symptoms  and positive findings of CA19-9, CT, MRI and PET-CT; (2). Unresectable LAPC defined by the criteria of National Comprehensive Cancer Network (NCCN) guidelines (Version 1. 2013); (3). MPC suffering from abdominal and/or back pain caused by primary lesions and/or metastatic regional nodes; (4). Receiving RT alone or as a part of multidisciplinary approach; and (5). Medical record and follow-up data completed.We found 69 consecutive patients matching the selection criteria from the medical record database in Fudan University Shanghai Cancer Center between May 2006 and April 2013. However, 63 patients were finally included in our analysis after excluding 6, who were lost to follow-up. Demographic and baseline characteristics, as well as treatment details for the entire group are summarized in Table CCRT was strongly recommended for all patients without contraindication, and RT alone, as an alternative for those who refused CCRT. Among 63 patients 36 (57.1%) patients received RT alone, and 27 (42.9%), CCRT. Before and/or after RT, systemic chemotherapy, regional intra-arterial chemotherapy (RIAC) and the combination of the above were administrated in 26 (41.3%), 39 (61.9%) and 15 (23.8%) patients, respectively.All patients received intensity modulated radiation therapy (IMRT). The patients were immobilized by vacuum-lock with no breath control devices. For LAPC the gross tumor volume (GTV) encompassed the primary tumor and metastatic lymph nodes, which were shown on CT scan, MRI or PET-CT. For MPC only symptom-causing lesions including the primary tumor and regional metastatic lymph nodes were irradiated. The planning target volume (PTV) was defined as the GTV plus 1.0 - 1.5 cm in the craniocaudal direction and 0.8 - 1.0 cm in all other directions. The median total dose delivered to PTV was 46.0 Gy  with a median fraction size of 1.8 Gy . The conventional fractionation (1.8 - 2.0 Gy/fx) was used in 60 patients and 30 Gy in 10 fractions, in 3 MPC patients. The organs at risk (OAR) included stomach, duodenum, kidney, liver and spinal cord. Their RT dosimetric parameters are listed in Table 2 on days 1, 8, 15, every 4 weeks) in 19 cases (70.4%), capecitabine  in 4 (14.8%), and S1  in 4 (14.8%). For the 26 patients underwent neoadjuvant and/or adjuvant chemotherapy, the regimens included GEM 1000 mg/m2 on days 1, 8, 15, every 4 weeks , GEM 1000 mg/m2 on days 1, 8 plus oxaliplatin 100 mg/m2 on day 1, every 3 weeks  and S1 40 mg/m2, twice daily on days 1–14, every 3 weeks . RIAC was given to 39 patients with a median cycle of 1 . Briefly, 5-Fr Rosch hepatic catheter was inserted via the femoral artery using Seldinger’s technique, and the catheter position was verified by digital subtraction angiography. The chemotherapy regimen was 1000 mg/m2 of GEM and 500 mg/m2 of 5-FU. Two-thirds of the dosage was injected via celiac artery and one-third, the superior mesenteric artery.Among the 27 patients who received CCRT, the chemotherapy regimens were gemcitabine (GEM)  version 3.0. The complete blood count and hepatic function was examined weekly during the treatment and on each follow-up visit after treatment completion. The pain was scored with the visual analogue scale (VAS), and was categorized into the none to mild (score 0–3), moderate (score 4–6) and severe pain (score 7–10). The serum CA19-9 and CEA was tested before and after treatment, and on each visit of the follow-up.p < 0.10) on univariate analysis along with another plausible covariate, tumor size, were entered in multivariate analysis using Cox’s proportional hazard model. All of the statistical analyses were performed using SPSS Statistics version 20.0 . P values less than 0.05 were considered statistically significant.The pain relief and OS were the primary endpoints. OS was counted from the date of diagnosis and estimated by Kaplan-Meier method. Univariate analysis was conducted by the log-rank test. Variables trending towards significance (p = 0.034) and thrombocytopenia (p = 0.019), and produced an increasing trend of elevated ALT (p = 0.051) compared to RT alone.Overall, the patients tolerated the treatments well. There were no treatment-related deaths. RT was terminated earlier than that planned in 3 patients (4.8%) when they had received 28.0 Gy, 30.0 Gy and 34.2 Gy, respectively due to the massive ascites resulting from tumor spreading in abdomen, refractory diarrhea or uncontrolled hyperglycemia. The observed RT-related and CCRT-related toxicities are listed in Table p = 0.000). Patients who had severe pain showed a greater amelioration than those had moderate pain, with the decreased score of 5.0 ± 1.5 and 3.4 ± 0.8, respectively (p = 0.000).Forty-four patients (12 LAPC and 32 MPC) presented abdominal and/or back pain prior to RT, which included moderate pain in 32 patients and severe pain in 12. Pain relieved after RT or CCRT in all of the 44 patients, with no pain to mild pain in 40, and moderate pain in 4. The mean VAS score before RT or CCRT was 5.9 ± 1.1, and significantly declined to 2.1 ± 0.9 after RT or CCRT  for all patients, and 14.2 months  for the alive or the censored. The median OS time for all 63 patients was 9.3 months , and 15.7 months  and 8.0 months , for LAPC and MPC patients respectively (p < 0.05), whereas neither tumor sizes (p = 0.232), nor RIAC (p = 0.507) showed significant associations with OS  and all positive findings by CA19-9, CT and PET-CT, which yielded 100% of positive predictive value according to our recent study (unpublished data).CCRT toxicity is the first one we have to consider. The data from a qualitative systematic review in 2009 demonstrated that CCRT increased OS when compared with exclusive RT, but was more toxic, in the management of LAPC [p = 0.002) with the median OS, 1-year and 2-year OS rates of 16.0 months, 58.6% and 33.5% for CCRT group, while 7.4 months, 30.6% and 11.1% for RT group. And by multivariate analysis, concurrent chemotherapy was confirmed as a beneficial factor for OS. These observations may further support the point of view that addition of concurrent chemotherapy to RT results in improved OS for patients with LAPC and MPC, with acceptable toxicities.As to the survival, a prospective randomized trial from Eastern Cooperative Oncology Group in 2011 reported that, for LAPC, the median OS, 1-year and 2-year OS rates were 11.1 months, 50% and 12% in patients treated with GEM-based chemoradiotherapy . A more p = 0.019). However, for our 31 MPC patients addition of RT did not seem to have any contribution to improvement of OS. A recent prospective phase 3 trial conducted in 48 centers reported that the median OS and 1-year OS rate was respectively 6.8 months and 20.6% for MPC patients treated with single-agent GEM, whereas it increased to 11.1 months and 48.4% when FOLFIRINOX regimen was used [For LAPC the outcome in CCRT was better than that in RT, the median OS, 1-year and 2-year OS rates being 19.5 months, 71.1% and 40.6% versus 7.4 months, 35.7% and 14.3%, respectively . Our investigation of RIAC was inconsistent with that reported in the literature . Howeverp = 0.003). Furthermore, as revealed by multivariate analysis, pretreatment CA19-9 ≥ 1000 U/mL was an independent indicator for poor OS.For prognostic predictors of pancreas cancers, CA19-9 has been identified as the best tumor marker. Two prior retrospective studies demonstrated that higher level of pretreatment CA19-9 with the cutoff value of 420 U/mL and 400 U/mL, respectively, was unfavorable predictor for OS in LAPC patients who underwent CCRT ,16. Alsop < 0.001) [p = 0.001) [p = 0.021). And tumors located in pancreas head was finally identified as an independent risk factor for poor prognosis by multivariate model.In the literature, the location of pancreatic cancer could be generally divided into head and body/tail cancers with their respective clinical manifestations and maybe, different malignant potential ,20. Ther< 0.001) . In cont= 0.001) . Our datIn conclusion, CCRT yielded a better OS than RT in LAPC and MPC patients, and moreover, was well tolerable. Pain relief was significant, particularly in patients with severe abdominal and back pain. Adding RIAC to RT or CCRT did not show any survival benefit. The favorable prognostic predictors were pancreas body/tail cancer, CA19-9 level of < 1000 U/mL at diagnosis and CCRT. Our study indicated that CCRT could be recommended to LAPC, and also MPC patients suffering from abdominal and/or back pain. RT is also a treatment option for those who could not tolerate, or refuse CCRT.


To examine whether elevated glycosylated hemoglobin A1c (HbA1c) is associated with hyperfiltration in a middle-aged and elderly Chinese population.th percentile.Anthropometric and biochemical examinations were performed in 2491 individuals from the general population, aged 40–79 years, who participated in the Huaian Diabetes Prevention Program. The estimated glomerular filtration rate (eGFR) was calculated from creatinine levels using the CKD-EPI formula. Hyperfiltration was defined as eGFR >90After adjustment , HbA1c and fasting plasma glucose (FPG) were found to be independently positively associated with eGFR. Additionally, after multivariate adjustment, the odds ratios (95 % CI) for hyperfiltration calculated for a 1-unit increase in HbA1c and FPG were 1.396 (1.089–1.790) and 1.306 (1.117–1.526), respectively. Compared with participants with HbA1c levels <5.7 %, the odds ratios (95 % CI) for hyperfiltration were 2.344 (1.025–5.364) in participants with HbA1c levels of 6.21–6.49 %, and 2.965 (1.537–5.720) in those with HbA1c levels ≥6.5 %.Elevated HbA1c (≥6.21 %) is associated with an increased odds of hyperfiltration in middle-aged and elderly Chinese. Longitudinal studies are needed to explore whether hyperfiltration increases the odds of diabetic nephropathy in individuals with prediabetes. With the continuing increase in the number of patients with diabetes mellitus, diabetic nephropathy (DN) has become the most common cause of end-stage renal disease (ESRD) in China , the USAGlycosylated hemoglobin (HbAlc) is widely accepted as being a good indicator of blood glucose control. In 2010, the American Diabetes Association (ADA) recommended that a HbAlc of 6.5 % or higher be used for the diagnosis of diabetes mellitus . In 2012Previous studies have indicated that patients with prediabetes  are at high risk of future T2DM and have impaired endothelial diastolic function . In addiRecent studies have focused on the correlation between HbA1c and the decrease in GFR in diabetes , 19. HowThis cross-sectional study was part of the Huaian Diabetes Prevention Program (ChiCTR-TRC-14005029) and was approved by the Huaian Second Hospital Ethics Committee, XuZhou Medical University, China. Written informed consent was obtained from all participants in this study.n = 358); (2) previously diagnosed diabetes mellitus (n = 528); (3) previously diagnosed renal disease, including autoimmune or drug-induced kidney disease, nephritis, renal fibrosis or renal failure, or previous kidney transplant with ongoing renal dialysis (n = 146); (4) previously diagnosed hepatic disease including fatty liver, liver cirrhosis and autoimmune hepatitis (n = 1132); (5) peripheral artery disease (n = 108); (6) coronary heart disease (CHD) including myocardial infarction and angina pectoris (n = 479); (7) any malignant disease (n = 8); or (8) eGFR <60 mL/min/1.73 m2 (n = 181). Therefore, 2491 subjects (1624 women) were deemed eligible for the analysis.In the present study, 5431 subjects (aged 40–79 years) attending annual routine health examinations under the auspices of the local governments between August and December 2014 at the health examination center of Huaian Second Hospital, Affiliated Hospital of Xuzhou Medical College in Huaian  were enrolled. Subjects were subsequently excluded from the analysis if any of the following criteria applied: (1) data were missing for calculation of the eGFR (2). Blood pressure (BP) was measured three consecutive times , and the mean value was used in the analysis. Venous blood samples were collected between 07:00 and 09:00, after overnight fasting, for the measurement of FPG, creatinine (CREA), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c). HbA1c was measured by high performance liquid chromatography . All quality control criteria of the ADA were applied for HbA1c measurement [Demographic characteristics, lifestyle information, and medical history were obtained by trained investigators using a standard questionnaire. Body mass index (BMI) was calculated as weight (kg) divided by height squared ; and mildly reduced eGFR as 60–90 mL/min/1.73 m2.eGFR was calculated from creatinine levels using the CKD-EPI formula . Normal The continuous variables in this study exhibited normal or approximately normal distributions, and are presented as means ± standard deviations (SDs). Categorical variables are presented as numbers (%). Comparisons between groups were made using the Student’s t test for continuous data and the chi-square test for categorical data.th percentile in the distribution of residuals from a multivariate linear regression analysis where we used absolute GFR values as the dependent variable and sex, age, weight, height, and use of ACE inhibitors or ARB as the independent adjusting variables. Then, 249 subjects (147 women) were defined as being with hyperfiltration, for a mean GFR of 102.27 (range 90.7–135.7) mL/min/1.73 m2.After verifying the assumption of a linear relationship between the dependent and independent variables that were introduced into a linear regression model , multivariate linear regression analysis was used to estimate the association of glucose metabolism type with eGFR. Identification of patients with hyperfiltration was done by selecting all participants >90Three models were constructed for each component of glucose metabolism. The first model was not adjusted. The second model was adjusted for age, gender, waistline, BMI, systolic blood pressure, and diastolic blood pressure. The third model was adjusted for age, gender, waistline, BMI, systolic BP, diastolic BP, cholesterol, log(triglyceride), HDL-c, LDL-c, serum uric acid, alcohol consumption, smoking status, sodium intake (6 g/day), the presence of hypertension (%), and the use of angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs).th, 75th, 90th and 95th percentiles as cut-off points. For HbA1c, the groups were: Q1, HbA1c <5.70 %; Q2, HbA1c 5.70–6.00 %; Q3, HbA1c 6.01–6.20 %; Q4, HbA1c 6.21–6.49 %; and Q5, HbA1c ≥6.5 %. For FPG, the groups were: Q1, FPG <5.40 mmol/L; Q2, FPG 5.40–5.70 mmol/L; Q3, FPG 5.71–6.30 mmol/L; Q4, FPG 6.31–6.99 mmol/L; and Q5, FPG ≥7.0 mmol/L). The associations of glucose metabolism  with the odds of hyperfiltration (as defined above) were estimated using multivariate logistic regression analysis in the same three models described above. P-values for the trends were calculated by Spearman correlation analysis of categorical variables and odds ratios (ORs) for the different groups, scored 0, 1, 2 and 3, respectively. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 16.0 .For analysis, the subjects were divided into five groups based on stratification of glucose metabolism levels using the 50P < 0.05), while HDL-c levels were significantly lower (P = 0.003). Interestingly, eGFR and the proportion of subjects with hyperfiltration were lower in the prediabetes group but higher in the T2NDM group. There was no significant difference between the three groups in systolic BP (SBP), diastolic BP (DBP), blood urea nitrogen (BUN), CREA, gender, and smoking status.The 2491 subjects (1624 women) included in the study were divided into three groups . As shown in Table 2.Among all subjects, 249 subjects (147 women) were defined as being with hyperfiltration, for a mean GFR of 102.27 (range 90.7–135.7) mL/min/1.73 mAs presented in Table P < 0.05).As shown in Table The present study has found that in a Chinese middle-aged and elderly population without a history of diabetes, HbA1c was positively associated with hyperfiltration independently of age, sex, BMI, waistline, BP, smoking status, alcohol consumption, hypertension status, and TC, TG, LDL-c and HDL-c levels. A similar association was found between FPG and hyperfiltration. However, since the two conditions often coexist, the results do not exclude the association of both acutely and chronically elevated glucose levels with eGFR, but cannot confirm an independent association. Furthermore, after multivariate adjustment, it was determined that the odds of hyperfiltration were significantly increased in patients with HbA1c ≥6.21 % and FPG ≥7.0 mmol/L. Subjects with HbA1c levels of 6.21–6.49 % had a 2.34-times higher odds of developing hyperfiltration than those with HbA1c <5.7 %. This suggests that hyperfiltration occurs not only in newly developed diabetes but also in prediabetes. Thus, more attention should be paid to subjects with HbA1c levels ≥6.21 % in order to maximize the prevention of hyperfiltration in patients without overt diabetes.Some studies, but not all, have shown an association between hyperfiltration and the subsequent development of nephropathy in subjects with diabetes . It is ith percentile as the definition of hyperfiltration, rather than an arbitrary threshold that has ranged in various studies from 125 to 140 mL/min/1.73 m2. Third, extensive covariate data, including age, gender, BMI, cigarette smoking status, alcohol consumption, sodium intake, hypertension history, SBP, DBP, log(TG), TC, HDL-c, LDL-c, FPG, SUA, eGFR, and waistline were used in the statistical analysis. The present study is supported by two previous ones [The present study differs from previous investigations of hyperfiltration , 22, 32.ous ones , but theous ones . Taken tCreatinine-based equations for estimating GFR include the Cockcroft-Gault equation proposed in 1976 , the ModThe etiology of increased GFR in the setting of elevated blood glucose and HbA1c is incompletely understood, but has been attributed to the effect of hyperglycemia on the renin-angiotensin-aldosterone system (RAAS). Miller et al. demonstrated that hyperfiltration responses to clamped hyperglycemia are related to intrarenal RAAS activation . MoreoveOf course, the present study has some limitations. First, the study sample might not be completely representative of the general population. Second, the methods used to measure eGFR may be influenced by non-GFR factors such as body composition and glycemic status , and by Elevated HbA1c levels are associated with increased odds of hyperfiltration in a middle-aged and elderly Chinese population. It is possible that hyperfiltration associated with elevated HbA1c (≥6.21 %) may be one of several mechanisms that cause renal injury in this population of patients.


Kenyaichthys and the new species †K. kipkechi sp. nov. The phylogenetic analysis unexpectedly places †Kenyaichthys gen. nov. in a sister relationship to the Rivulidae , a probable explanation might be lack of available synapomorphies for the Rivulidae, Nothobranchiidae and Aplocheilidae. The specimens of †K. kipkechi sp. nov. show several polymorphic characters and large overlap in meristic traits, which justifies their interpretation as a species flock in statu nascendi. Patterns of variation in neural and haemal spine dimensions in the caudal vertebrae of †Kenyaichthys gen. nov. and the extant species studied indicate that some previously suggested synapomorphies of the Cyprinodontoidei and Aplocheiloidei need to be revised.The extant Cyprinodontiformes (killifishes) with their two suborders Cyprinodontoidei and Aplocheiloidei represent a diverse and well-studied group of fishes. However, their fossil record is comparatively sparse and has so far yielded members of the Cyprinodontoidei only. Here we report on cyprinodontiform fossils from the upper Miocene Lukeino Formation in the Tugen Hills of the Central Rift Valley of Kenya, which represent the first fossil record of an aplocheiloid killifish. A total of 169 specimens - mostly extraordinarily well preserved - and a sample of ten extant cyprinodontiform species were studied on the basis of morphometrics, meristics and osteology. A phylogenetic analysis using PAUP was also conducted for the fossils. Both the osteological data and the phylogenetic analysis provide strong evidence for the assignment of the fossils to the Aplocheiloidei, and justify the definition of the new family †Kenyaichthyidae, the new genus † The extant order Cyprinodontiformes contains about 1,120 species  the ordeProlebias SAUVAGE, 1874, from the Oligocene and Miocene of Europe and Asia, which has recently been identified as a paraphylum and now includes several additional genera : 1142´04, 1144/1146´04, 1145´04, 1147´04, 1148(1)´04, 1149´04, 1150´04, 1151/1152´04, 1153´04, 1154a/b´04, 1155´04, 1156´04, 1157(1)/1158(1)´04, 1159a(1)/b(1)´04, 1159a(2)/b(2)´04, 1160a/1161b´04, 1160b/1161a´04, 1162´04, 1163a(1)/b(2)´04, 1163a(2)/b(1)´04, 1164a/b´04, 1165a/b´04, 1166a´04, 1166b´04, 1167´04, 1168´04, 1170´04, 1171´04, 1172´04,1174´04, 1175´04, 1176a/b´04, 1177´04, 1178(1)´04, 1180(1)´04, 1181(1)´04, 1181(2)/1183(1)´04, 1182´04, 1184(1)´04, 1185/1186´04, 1187´04–1189´04, 1190´04, 1192a/b´05, 1192´04, 1193´04, 1194´04, 1198a/b´04, 1199a/b´04, 1200´04, 1202´04, 1203a/b´05, 1204´05, 1204´04, 1206(1)/1211´04, 1209´04, 1213(1)´04, 1215(1)´04, 1217a(1)/b(1)´04, 1218´04, 1218a/b´05,1219(1)´04, 1220(1)´04, 1220R´04, 1221(1)´04, 1227(1)´04, 1228(1)/1237R(1)´04, 1233/1234(1)´04, 1234(2)´04, 1234R´04, 1236(1)´04, 1237(1)´04, 1237(2)´04, 1324´04, 1325´04. In addition, 92 further specimens are tentatively assigned as †tion see .Late Miocene, about 5.7–6 Ma.Tugen Hills, Inoswa Kamelon (72 specimens) and Koibochepkweny (5 specimens), Lukeino Formation, Kenya.Named for Joseph Kipkech, Kenya, in recognition of his long-time devoted commitment to the development of education and science in Baringo County.As for the family.Small fishes, ranging in size from 22 to 40 mm total length (TL), and 20 to 36 mm standard length (SL) see . The snoThe frontals are large and rectangular . The parThe shape of the dentary is elongate; its upper limb is probably as deep as the lower limb . A singlThe toothless maxilla is long and slender and has three tiny processes at its anterior end. The first of these is anteriorly directed and represents the dorsal process; the two other structures probably comprise the ventral process .The premaxilla and maxilla are of nearly equal length  posteriorly Figs . NotablyThe gill arches are not clearly recognizable, but one specimen (1212a/b´04) does show a pharyngobranchial tooth-plate that bears multiple rows of molariform teeth.In 50% of the specimens, where the vertebral column is preserved until the end of the dorsal fin, the vertebral column is straight to slightly curved, whereas in the remainder, the abdominal part of the vertebral column displays a hunchback-like curve . The totThe neural and haemal spines of the anterior caudal vertebrae are upright, whereas those of the following ones are posteriorly inclined. All spines of the caudal vertebrae are long and almost reach the dorsal  border of the body  is recognizable in 90 specimens; 49 of them possess five preural vertebrae, the remainder have six see . The preThe uroneural  is usually not visible, but in some specimens it is recognizable as a short and tiny structure closely attached to the proximal portion of the upper segment of the hypural plate and terminal centrum ; lateralSupernumerary neural and/ or haemal spines can be observed in 32 out of 127 specimens in which neural and/ or haemal spines are visible in the caudal region see . The addThe proximal part of the parhypural is recognizable in 78 specimens and is polymorphic, i.e. it may either be reduced and lack contact with the terminal centrum and hypural plate . 55 specimens show an epural without an anterodorsal projection; the epural is curved in 35, and straight in 20 of these specimens. The 27 remaining specimens display a straight (19 specimens) or curved (eight specimens) epural with an anterodorsal projection. Additional six specimens have the caudal skeleton preserved, but the orientation of the specimen is unclear see .Both the neural spine PU2/PU4 and PU2/PU5 ratios were >1.0 in most specimens . The haeCycloid scales  are visi7% (n = 11) of the specimens show a regularly distributed granulation-like structure between the spines and rays of all fins, around the entire vertebral column, on individual vertebrae, and sometimes also on the head . Most liThe fossil specimens show variation in the length and width of the hypural plate, in the numbers of preural vertebrae, and also in the width of the haemal and neural spines of the preural vertebrae. In order to understand the taxonomic meaning of this variation, we therefore asked whether these characters show a similar tendency to vary in extant killifish.Aphanius  of A. farsicus, three females  and three males (2 c&s and 1 xr) of A. arakensis, and two females and two males  of A. mesopotamicus were analysed with regard to the hypural plate dimensions in the two sexes , a total of 10 specimens of the Nothobranchiidae , and a total of 29 specimens of the Cyprinodontidae . We found intraspecific variation of the preural vertebrae number in N. orthonotus . The remaining specimens consistently displayed four preural vertebrae  ;a synapomorphy for Cyprinodontoidei vs. Aplocheiloidei ;a synapomorphy for Nothobranchiidae/Rivulidae vs. all other cyprinodontiform families .In the case of (iii), however, it is not clear from previous studies how “slightly wider” and “distinctively wider” should be defined. We consider here ratios of >1.0 and <2.0 as slightly wider and ratios of ≥2.0 as distinctively wider.We used the comparative material described above to verify the phylogenetic significance of these characters. Ratios between spines were calculated based on the maximal width of the respective spine see .Aphanius mesopotamicus, which reveals the neural spine of PU2 as wide as the neural spine of PU4.The neural spine of PU2 was wider than the neural spines of PU4 and PU5 in almost all specimens studied , as expeThe neural and haemal spines of PU3 were wider than those of PU5 in the cyprinodontoid specimens Tables . HoweverP. playfairii . On the other hand, the PU2/PU5 mean value for the nothobranchiid species is significantly smaller than in the studied cyprinodontoid species , as expected from literature data.In seven of the ten studied extant nothobranchiid specimens, the haemal spine of PU2 is distinctively wider (ratio ≥2.0) than those of PU4. Moreover, six of the ten specimens show a ratio of ≥2.0 for PU2/PU5. However, only the character state “slightly wider” (1.0< ratio <2.0) is expected for Nothobranchiidae and Rivulidae from previous work , like the studied extant cyprinodontiform species, with the exception of one specimen of Aphanius mesopotamicus , but the range of the PU3/PU5 ratios is comparatively close to that of the studied aplocheiloid specimens , but also not as sharply bent as is typical for the Cyprinodontoidei .Moreover, †ate; see  page 542ate; see  page 544Kenyaichthys is the presence of five or six preural vertebrae, whereas Aplocheiloidei and Cyprinodontoidei usually possess four or five preural vertebrae. Six preural vertebrae have only been reported for three distantly related cyprinodontoids, i.e. the anablepid Anableps, the cyprinodontid Orestias and the fundulid Fundulus , , , 50], a, a50], a(DOC)Click here for additional data file.S10 Table(DOC)Click here for additional data file.S1 NEXUS File(NEX)Click here for additional data file.S1 FigA on a rounded tip; B on a blunt tip; C on spines with tip not covered by caudal fin rays and on spine covered by fin rays.(TIF)Click here for additional data file.


AbstractNational Marine Fisheries Service laboratory in Pascagoula, Mississippi. This sampling has resulted in numerous new species of kinorhynchs from the shelf sediment, two of which are described in detail in this paper. Other species descriptions from this research effort include Echinoderesaugustae, Echinoderesskipperae, and Echinoderescharlotteae. We now describe Echinoderesromanoisp. n. and Echinoderesjoyceaesp. n., which are unique in their spine, tube, and glandular cell outlet patterns.Meiofauna sampling on the continental shelf of the northern Gulf of Mexico has been ongoing since 2007, on annual cruises in collaboration with the  Kinorhyncha has been underreported from the Gulf of Mexico, though recently new investigations are adding to our knowledge of the Gulf species. Currently there are few kinorhynchs identified to species from the Gulf, though their presence and abundance are well documented. The known species PageBreakdiversity in the Gulf of Mexico includes Echinoderessteineri , EchinoderescoulliEchinoderesremanei , Kinorhynchuslangi , Campyloderescf.vanhoeffeni Zelinka, 1913, CentroderesbarbanigraCentroderescf.drakeiEchinoderesskipperae Sørensen & Landers, 2014, Echinoderesaugustae Sørensen & Landers, 2014, Echinoderesbookhouti Higgins, 1964 and EchinoderescharlotteaeNational Marine Fisheries Service (NMFS) lab in Pascagoula, Mississippi. This collaboration began in 2007 and continues currently, with sediment collection occurring on annual fall cruises. The first two reports from the kinorhynch analysis of this long term meiofauna study described three new species, Echinoderesaugustae, Echinoderesskipperae, and Echinoderescharlotteae, and also provided a redescription of Echinoderesbookhouti  or an Olympus BX51 (University of Copenhagen) light microscope equipped with Nomarski interference contrast optics using digital cameras. Line art illustrations were based on mounted specimens that were drawn using Adobe Illustrator® CS6 or Adobe Photoshop Elements® software. Measurements were made with CellSens® software. All dimensions reported in the tables are based on LM measurements. All light microscopy type material is deposited at the MuseumNatural History  of Denmark .The samples were fixed immediately in 5–10% formalin on the cruise, and the meiofauna was subsequently extracted by Ludox centrifugation . After sPageBreakUniversity Research Instrumentation Facility . Specimens stored in 70% isopropanol were hydrated, post-fixed in OsO4 vapor, then dehydrated to 100% ethanol through a graded series, critical point dried, mounted on aluminum stubs, and sputter-coated with gold. Specimens were photographed with a Zeiss EVO 50 SEM, using the backscatter and secondary electron detectors.Specimens for scanning electron microscopy were observed at the Auburn PageBreakTaxon classificationAnimaliaEchinorhagataEchinoderidaehttp://zoobank.org/E6387ED6-A68B-4DB2-A835-777A585D5DECZMUC KIN-962), collected from sediment on November 13, 2014, at station 012-2014 , mounted in Fluoromount G®, deposited at the MuseumNatural History  of Denmark. Paratypes include two females (ZMUC KIN-963 and KIN-964) from station 012-2014, two females (ZMUC KIN-965 and KIN-966) from station 19-2014, one male from station 154-2010 (ZMUC KIN-967) and one male from 050-2013 (ZMUC KIN-899). All paratypes are mounted in Fluoromount G® and deposited at the MuseumNatural History  of Denmark. Additional nontype material is listed in Table Holotype: Adult male , 5, and 8. This combination of spines, tubes, and glandular cell outlets is unique among all species in the genus. The spine/tube arrangement is not unusual among congeners: 36 additional species share the presence of middorsal spines on segments 4 to 8 and lateroventral tubes/spines on segments 5 to 9, and out of these, ten also lack tubes on segment 2 as PageBreakdoes Echinoderesromanoi sp. n.: EchinoderesangustusEchinoderesaquiloniusEchinoderestubilakEchinoderesremanei  and placid length (10 µm) of Echinoderesromanoi n. sp. . The same distinction is true for Echinoderesremanei, redescribed by Echinoderesbrevicaudatus has lateral dorsal tubes on segment 10, and short stubby lateral terminal spines, distinct from the new species. Echinoderescernunnos has glandular cell outlets type 2 located similarly to Echinoderesromanoi on segments 2, 5 and 8, though Echinoderescernunnos also has glandular cell outlets type 2 in the midlateral position on segment 7 and additionally has elongated spinous tergal extensions. Echinodereskoreanus has spines in the lateral dorsal positions on segments 7 and 8, and tubes in the laterodorsal position on segment 10, unlike the new species. Echinoderesstockmani is distinguished by having the lateral spines on segment 8 distinctly longer than those PageBreakPageBreakPageBreakon segment 9, unlike Echinoderesromanoi n. sp. Echinoderesobtuspinosus has glandular cell outlets type 2 similarly to Echinoderesromanoi on segments 2 and 8. However, Echinoderesobtuspinosus has glandular cell outlets type 2 in the subdorsal position on segment 4 and none on segment 5. Further, Echinoderesobtuspinosus has short stubby lateral terminal spines. Finally, Echinoderesbookhouti has lateral accessory spines on segment 8, and lacks glandular cell outlets type two on segment 5 and in the laterodorsal and sublateral position on segment 2.remanei , Echinodns, 1964 , 2016. MTaxon classificationAnimaliaEchinorhagataEchinoderidaehttp://zoobank.org/B7ED6634-BA3F-4250-84DC-DE7CBC53A19DZMUC KIN-845), collected from muddy sediment on October 16, 2010, at station 010-2010 , mounted in Fluoromount G®, deposited at the MuseumNatural History  of Denmark. Paratypes include one female (ZMUC KIN-849), collected at same time and locality as the holotype, and three males, collected at stations 016-2010 (ZMUC KIN-850), 068-2012 (ZMUC KIN-922), and 031-2013 (ZMUC KIN-867). All paratypes were mounted in Fluoromount G® and deposited at the MuseumNatural History  of Denmark. Additional non-type material includes one female from station 033-2014. See Fig. Holotype: Adult male (Echinoderes (183–209 µm) with middorsal spines on segments 4, 6 and 8, and spines in lateroventral positions on segments 6 to 9. Tubes present in ventrolateral positions on segment 2, in lateroventral positions on segment 5, and in laterodorsal positions near the posterior margin of segment 10. Glandular cell outlets type 2 present in subdorsal position on segment 2, in midlateral position on segment 6, and in sublateral position on segment 8.Conspicuously small Adults conspicuously small (183–209 µm in trunk length), with head, neck and eleven trunk segments Figs –7. For cThe head Fig.  consistsPageBreakPageBreaknarrower, measuring 6 µm in width at their bases. The trichoscalid plates, each with a trichoscalid, present in subdorsal, laterodorsal and ventromedial positions.The neck Figs , 6A–D haSegment 1 Figs  consistsPageBreakthickness and interrupted in subdorsal and ventrolateral positions. Glandular cell outlets type 2 are located in subdorsal position. Secondary pectinate fringe not detected on this or any of the following segments. Bracteate cuticular hairs evenly distributed in a medial band around the segment. The posterior segment margin is straight and consists of a pectinate fringe, with fringe tips alternating with small trichoid extensions.Segment 2 Fig.  consistsSegment 3 Figs , and remSegment 4 Figs  with aciSegment 5 Figs  with tubPageBreaktion. Glandular cell outlets type 2 present in midlateral position. Pachycycli, pectinate fringe of posterior margin and cuticular hairs as on preceding segment.Segment 6 Figs , 7A, D–FSegment 7 Figs  with aciSegment 8 Figs  with midSegment 9 Figs , 7H, I wSegment 10 Figs , 7H, I wSegment 11 Fig. , 7H, I wThis species is named after Joyce Wright Landers—the wife of the first author.Echinoderesjoyceae sp. n. is characterized by the presence of middorsal spines on segments 4, 6 and 8, ventrolateral tubes on segment 2, lateroventral tubes/PageBreakPageBreakPageBreakspines on segments 5 to 9, and laterodorsal tubes on segment 10. This combination of spines and tubes is not unusual among congeners, and is shared with five other species: EchinoderesbermudensisEchinodereskristenseniEchinoderesabbreviatusEchinodereshispanicusEchinoderesintermedius Sørensen, 2006. (Echinoderesjoyceae using a number of characteristics. All five of these species have tergal extensions on segment 11 distinct from Echinoderesjoyceae. Additionally, the new species has a distinctive distribution of glandular cell outlets type 2, with locations in the subdorsal position on segment 2. Amongst the abovementioned species this is only found in Echinodereskristenseni, which also has glandular cell outlets type 2 in lateroventral positions of segment 2, not present in Echinoderesjoyceae sp. n. The most unique character combination in Echinoderesjoyceae sp. n. though, is the presence of glandular cell outlets type 2 in midlateral positions of segment 6 and in sublateral positions of segment 8. This combination is not found in any other species of Echinoderes. Furthermore, Echinoderesjoyceae sp. n. is characteristic by its minute size. With a trunk length ranging from 183 to 209 µm, Echinoderesjoyceae sp. n. is among the smallest known Echinoderes.n, 2006. . These sEchinoderesromanoi sp. n. and Echinoderesjoyceae sp. n., and reports the known distribution of the two new species along the northern Gulf of Mexico continental shelf. In common with the previous shelf species reported so far during this long term meiofauna sampling is the broad distribution of the taxa. Echinoderesromanoi sp. n. and Echinoderesjoyceae sp. n. are distributed across wide regions of the United States’ Gulf shelf, with the location of Echinoderesromanoi extending from central Louisiana to western Florida, and with Echinoderesjoyceae sp. n. extending from Texas to Florida. Similarly, Echinoderesaugustae, Echinoderesskipperae, Echinoderescharlotteae, and Echinoderesbookhouti (Echinoderes species across the Gulf, it is interesting that they have not been observed in coastal marshes. In a recent study on the effects of the Deepwater Horizon oil spill from 2010 in Barataria Bay, Louisiana (Echinoderescoulli, with no offshore species present (identifications in the Echinoderescoulli is an estuarine species, which has not been observed in our offshore samples. Our sampling on the continental shelf has consistently sampled sediment from high salinity waters, yielding species that may not tolerate estuarine conditions. Sampling along the U.S. shoreline in high and low salinity waters are needed to determine if the shelf species are found in the intertidal zone or marshes.This study describes ookhouti , all repouisiana , all idePageBreak


The purpose of this study was to compare the refractive outcomes of small incision lenticule extraction (SMILE) in high-myopic patients with those of mild- to moderate-myopic patients.This study included 183 eyes of 92 myopic patients treated with SMILE using a VisuMax 500-kHz femtosecond laser. Treated eyes were divided into two groups, according to the preoperative spherical equivalent (SE): mild to moderate myopia  and high myopia . Follow-up visits were at 1 day, 1 week, and 1, 3, 6, and 12 months. The outcome measures included uncorrected distance visual acuity (UDVA), best-corrected distance visual acuity (BDVA), postoperative SE, efficacy index, safety index, and predictability.p = 0.18). At 12 months postoperatively, 93.1 % and 76.8 % had an UDVA of 20/20 or better in the A and B groups, respectively. In the A group, 87.9 % and 96.6 % were within ±0.5 D and ±1.0 D, respectively, of the intended correction; in the B group, 88.0 % and 97.6 % were within ±0.5 D and ±1.0 D, respectively. The efficacy index was 1.04 ± 0.19 in the A group and 0.99 ± 0.19 in the B group. The safety index was 1.27 ± 0.17 for the A group and 1.24 ± 0.17 for the B group. The efficacy and safety index were not significantly different between the two groups 12 months postoperatively .Preoperative SE was −5.05 ± 0.71 D in the A group and −7.67 ± 1.01 D in the B group. No differences were observed between −0.13 ± 0.38 D in the A group and −0.24 ± 0.35 D in the B group 12 months postoperatively (This study showed that SMILE is effective and safe for correcting high myopia, as well as mild to moderate myopia. Small incision lenticule extraction (SMILE) is a flap-free refractive surgery, in which the corneal stromal lenticule is cut by a femtosecond laser and removed through a small corneal incision tunnel , 2. ReceHowever, complications after surgery can include dry eyes , cornealThere have been numerous studies , 12–14 oThis retrospective study evaluated the outcome of consecutively treated patients with SMILE at the Onnuri Eye Clinic in Jeonju, Korea from May 2012 to May 2014. The analysis included all eyes with a 12-month follow-up, spherical myopia less than −12.0 D and myopic astigmatism less than −4.0 D cyl, a minimum age of 18 years, corrected distance visual acuity (CDVA) of 20/40 or better (>0.3 logMAR), and a minimum calculated postoperative residual stromal bed of 250 μm. The study included 183 eyes from 92 patients with myopia (with and without astigmatism) who fulfilled the criteria specified above. The study was approved by the Institutional Review Board/Ethics Committee of Yeouido St. Mary’s Hospital, Seoul, Korea and performed in accordance with the tenets of the Declaration of Helsinki. The treated eyes were divided into two groups: mild- to moderate-myopia patients  and high-myopia patients . The two groups were compared with respect to the efficacy, predictability, and safety of the surgery. The efficacy index /preoperative UDVA’) and the safety index (defined as ‘postoperative CDVA/preoperative CDVA’) were estimated.Patients underwent a thorough ophthalmic examination that included autokeratometry, autorefractometry, intraocular pressure tonometry , pupillometry , corneal tomography, corneal thickness measurements , measurement of the UDVA and CDVA, measurement of manifest and cycloplegic clinical refraction, slit-lamp evaluation, and fundoscopy.The same surgeon (CYT) performed all surgical procedures. A VisuMax 500-kHz femtosecond laser  was used for the SMILE treatment . The lenticule diameter was 6.5 mm and the cap diameter was 7.5 mm. The incision length varied from 2.0–2.5 mm, and the intended cap thickness was 100–120 μm. The SMILE procedure has been described previously. After surgery, all patients received a topical antibiotic for 5 days and a topical steroid for 2 weeks. Hyaluronic acid lubricating drops were prescribed for 2 weeks (minimum).All patients were routinely examined at 1 day, 1 week, 1 month, 3 months, 6 months, and 12 months. At each visit, CDVA, UDVA, objective and manifest refractions, keratometry, intraocular pressure, corneal topography, and slit-lamp examination were performed. All postoperative complications were noted.P-values less than 0.05 indicated statistical significance.Statistical analyses were performed using the SPSS software . Graphics were generated using Microsoft Excel 2007 . All values are given as the mean ± standard deviation. Statistical analyses for visual acuity were based on logMAR units. Student’s t-test was used to compare the two groups. p = 0.02). The mean preoperative spherical equivalent was −5.05 ± 0.71 D and −7.67 ± 1.01 D for the A and B groups, respectively (p = 0.000). The central cornea thickness of the B group (526 ± 31.5 μm) was thicker than that of the A group (510.5 ± 29.9 μm) (p = 0.002). The expected residual corneal bed of the A group (301.0 ± 28.0 μm) was thicker than that of the B group (291.1 ± 26 μm) (p = 0.025). No significant difference was evident between the two groups with respect to age, mean corneal power, or cylinder astigmatism  in all eyes. The mean preoperative CDVA was −0.069 ± 0.047 logMAR and −0.045 ± 0.052 logMAR for the A and B groups, respectively .At 1 day, 1 week, 1 month, 3 months, 6 months, and 12 months, 65.5, 81.9, 86.8, 87.5, 89.9, and 93.1 % of eyes in group A and 63.2, 79.2, 78.8, 80.8, 78.8, and 78.4 % of eyes in group B, respectively, had an UDVA of 20/20 or better. There were significant differences between two groups in 1 month and 12 months .The efficacy index was 1.04 ± 0.14 and 0.99 ± 0.14 for A and B groups, respectively. There was no significant difference between the two groups .The safety index were 1.27 ± 0.17 in group A and 1.24 ± 0.17 in group B. No significant difference was evident between the two groups (Additionally, no visually threatening complications occurred in either group. There were no cases of epithelial ingrowth, severe diffuse lamellar keratitis, or keratoectasia in either group.There is growing interest in SMILE as a new alternative refractory surgery. Although multiple studies , 12–14 hThere was no difference in both CDVA and UDVA between the two groups through the 1-year follow-up period. This is consistent with previous study of Hjortdal et al.  which suFor Groups A and B, 93.1 % and 76.8 % of eyes, respectively, had 20/20 or better UDVA at 12 months. According to previous studies, 88 % in Sekundo et al. , 83 % inIn the predictability evaluation, no significant difference in the rates within ±0.5 D and ±1.0 D was evident between Groups A and B; these results are in good agreement with previous results –17 of 85In the safety evaluation, there was no significant difference in the safety index at 12 months; 3–4 % had one line loss in both groups, which is comparable to the results from previous studies –17 indicIn this study, no myopic regression was observed from 1 week to 12 months in either group, which is compatible with previous studies. Although Sekundo et al.  observedIn the study population, the preoperative central cornea thickness and preoperative CDVA differed significantly between the two groups, which might affect the long-term outcomes after surgery. This is a potential limitation of this study.Ivarsen et al.  determinComparing the results at 6 and 12 months in this study, there were no significant differences in efficacy, predictability and safety. This is compatible with the results of previous studies that reported similar 6-  and 12-mIn this study, the clinical SMILE outcome of high-myopia patients, including efficacy, predictability, and safety, did not differ from that of mild- to moderate-myopia patients; however, it is noteworthy that the mean preoperative SE was −7.70 D and the mean expected residual corneal bed thickness was 291.1 μm in the high-myopia patients. The results of this study suggest that SMILE surgery could be useful refractive surgery in high-myopia patients with a relatively thin corneal thickness.SMILE is effective and safe for correcting high myopia, as well as mild to moderate myopia.


The per capita estimated for the wastewater production and treatment were determined to be 186.06 ± 7.85 and 136.72 ± 5.43 L/d.cap, respectively. It is estimated that about 504 m3/d and 346 m3/d of sludge, will be produced and waste as excrement raw sludge, respectively, in Tehran. Simple regression models were presented the relationships such as the change of collected and treated wastewater with population and changes of GPCD parameters with each other. It was revealed that the Tehran’s wastewater may be classified as highly degradable, but during recent decades its Biodegradability Index (BI) has been reduced up to 15%. The new suggested revised per capita parameters can be used for design purposes in Tehran, and possibly, in areas with similar characteristics, substituting the classical values obtained from foreign textbooks. These values could help in designing more accurate treatment systems and may lower the required capacity for the treatment of wastewater up to 40% in Tehran.The amount of wastewater characteristics loading is one of the main parameters in the design of wastewater collection and treatment systems. The generation per capita per day (GPCD) of wastewater characteristics was estimated by analyzing the monthly data of nine wastewater treatment plants in Tehran, capital city of Iran. GPCD values were calculated from measured collected wastewater flow, the population and concentration data. The results indicated the values of 32.96 ± 1.91, 49.25 ± 2.49, 37.31 ± 2.44, 6.77 ± 0.53, 1.96 ± 0.11, 92.23 ± 5.68, 2.07 ± 0.39 and 128.96 ± 6.69 g/d.cap of GPCD for BOD The per capita loading of wastewater characteristics such as chemical oxygen demand (COD), Biological oxygen demand  in Tehran those can treat more than 100 MCM of wastewater per year . By cons5, COD, TSS, TKN, P, TDS, ON(Organic Nitrogen) and TS) generation per capita per day (GPCD) according to the recorded data of nine public wastewater treatment systems in Tehran. The per capita loading of wastewater is an important parameter in the design process of a treatment plant’s units. Iran’s water and sewage utility (ABFA company) states that per capita water consumption in Tehran is currently about 378 liters per day [One of aims of the study reported in this paper is to re-establish the main wastewater pollution measures (including BOD per day . Our stu5), Total Dissolved Solid (TDS), Total Solids (TS), Total Suspended Solid (TSS), Chemical Oxygen Demand (COD), Organic Nitrogen (ON), Total Phosphorous (TP) and Total Kjeldahl Nitrogen (TKN). Data analysis involved the data pre-processing and conducting some descriptive and analytical studies using Microsoft Excel 2007 and R software packages.The data were used in this study obtained from monthly reports (from 2007 to 2013) of nine wastewater treatment plants in Tehran including Sahebgharaniyeh, Mahallti, Zargandeh, Qeitariyeh, Qods, Shush, Ekbatan, Dowlatabadi and Jonoob. The data includes the quantitative information of wastewater production and characteristics data over 80 months from April 2007 to November 2013 for all domestic WWTPs in Tehran. The analysis of wastewater samples were done by the laboratory stuffs of WWTPs according to Standard Methods for the Examination of Water and Wastewater . The parThe study included data pre-processing and preparing them to make the estimates of desired parameters. Initial data processing, although is a time consuming step, but is very important part in the success of statistical analysis . At thisThe descriptive statistics of raw (720 instances) and pre-processed (499 instances) data are summarized in Tables The estimation of pollutant generation per capita per day (GCPD) values was conducted according to the following equations:The pollutant discharge per capita  with the wastewater treatment system was defined and determined in terms of pollutant generation per capita  and pollutant removal efficiency  of the wastewater treatment systems as follow :\documen3/year estimated collected wastewater from 2007 to 2013, only 19,749,770 m3/year of it were treated. In other words, about three MCM per year (13% of estimated collected wastewater from 2007 to 2013) were discharged into the environment without adequate treatment. The mathematical relationships between wastewater flows and population in Tehran were shown in Figure The total yearly averaged population covered by Tehran’s nine municipal wastewater treatment plants from 2007 to 2013 was determined to be 4,502,065 persons per year. In average, out of 22,778,632 m3/d had been allocated for the treatment of wastewater produced by 3,150,000 persons up to June 2014. This means that the per capita loading of wastewater considered by designers for the design of a wastewater treatment plant in Tehran, is about 215 L/d. By a roughly estimation according to Figure 3/d is a sufficient capacity that needs to be allocated. This capacity is about 40% lower than current allocated value. Therefore, the overestimated per capita loading value of 215 L/d in Tehran, leads the system to be designed for a capacity more than that is required.According to the latest report of the basic operational items of Tehran’s wastewater company , a capacThe pattern changes of Tehran wastewater quality contents from 1984 to 2013 is another issue were assessed in Table 5, COD, TSS, TKN, P, TDS, ON and TS, respectively. In a study conducted by Azimi and Ameri [5, COD, TSS, TKN and P, respectively. Although their estimated values are not representative of all treatment plants, comparison of them with the GPCD estimated in our study show that the GPCD of BOD5, TKN, and P were not considerably changed, but it was decreased by 71% and 79% of estimated values (in 1997) for TSS and COD parameters, respectively. This result is consistent with the pattern of TSS changes in Table The estimated per capita loadings for domestic wastewater of Tehran, as summarized from the results of this investigation, are presented in Table nd Ameri  on the e5/COD ratio, is called Biodegradability Index (BI), was determined. The BI Index varies from 0.4 to 0.8 for domestic wastewaters. If BOD5/COD is > 0.6 then the waste is fairly biodegradable and can be effectively treated biologically. If BOD5/COD ratio is between 0.3 and 0.6, then seeding is required to treat it biologically. If BOD5/COD is < 0.3 then it cannot be treated biologically [For evaluating the biodegradability of Tehran’s domestic wastewater the BODogically ,22. Fromogically . Many deogically . This maCOD and GCPDBOD (R2 = 0.74). The next relationship with high correlation (R2 = 0.66) was found between GCPDBOD and GCPDTSS. These relationships may be useful for designers to help them to estimate the GPCD values from each other.Table PerP) was determined to be 186.06 ± 7.85 while the CollPerP of 199.67 L/d.cap was estimated from the study’s results of Sharifi Sistani [The per capita collected wastewater , were more than those for 2013 obtained to be 28.9, 32.3, 6.1 and 1.6 g/d.cap, respectively. The result indicates and confirms that the GCPD of TSS considerably reduced during the recent decade.As shown in Table 5 and 50 to 60 g/d.cap of total suspended solids (TSS). These values have been universally used in the design of wastewater treatment systems and unchallenged since the publication of this standard in Iran. The recommended values of 40-50 g/d.cap for BOD5 and 50-60 g/d.cap for TSS are more than the values of 31.05-34.86 and 34.87-39.76 g/d.cap estimated in this study, respectively. In another word, the estimated values of GCPD for BOD5 and TSS for Tehran’s wastewater are 27% and 32% lower than the mean of recommended values by Department of Energy, respectively. Using the guideline values recommended by Department of Energy for the design of wastewater treatment plant for the city of Tehran, with different living habits in different parts of the city, may results in considerable overestimations in the design process of new treatment plants. Thus, it is recommended that our estimated GPCDs be used in the design of new wastewater treatment plants in Tehran instead of recommended values by Department of Energy.According to the technical criteria standard N. 3-129 , a new s5 and TSS [In Table  and TSS .Table 7It is estimated that over 90% of the treated wastewater effluent from treatment plants across Iran country is reused in some way; however, much of it is mixed with freshwater before further use, particularly in the suburban areas . The dir5, 89.65–90.97% for COD, 91.48–93.18% for TSS, 28.65-31.72% for TS, 1.73-2.76% for organic nitrogen (ON), 58.52-61.24% for P and 84.62-88.26% for TKN.The PDC with the wastewater treatment systems in Tehran was defined and determined in terms of GCPD and pollutant removal efficiency . The pollutants removal efficiency of wastewater treatment systems in Tehran ranged 92.11–93.41% for BOD5, COD, ON and TP of the United States wastewater treatment systems were almost thrice, four times, nine times and twice as those in Tehran, respectively. Conventional and nutrient removal activated sludge treatment systems are regarded as representative wastewater treatment processes in urban area of developed countries such as USA. PDCs with these treatment systems were estimated as 16.1 g-BOD5/d.cap, 31.6 g-COD/d.cap, 4.9 g-ON/d.cap, and 3.2 g-TP/d.cap [5/d.cap, 4.46 ± 0.34 g-COD/d.cap, 1.82 ± 0.32 g-ON/d.cap, and 0.76 ± 0.04 g-TP/d.cap. Deployment of larger removal efficiency treatment systems will decrease PDC with increase of PRE. When judging only from pollutant discharge reduction function of treatment systems, countries or cities with smaller GCPD and larger PDC-BOD5 should be prioritized for investments on wastewater treatment measurements including assistances in order to improve ambient water quality [The ranges of estimated PDC summarized in Table TP/d.cap  while PD5 discharge reduction from domestic wastewater, respectively .The estimated PDC of some regions studied by Tsuzuki  were comly Table . The PDC5-PDC in sewage treatment plants of Tehran may results from the fact that the per capita loading of BOD5 in Tehran is lower than that for other countries and on the other hand the removal efficiency of BOD5 in most of treatment plants are more than 90%.The comparatively low BOD3/d and 346 m3/d of sludge will be produced and waste as excrement raw sludge, respectively, in Tehran. This high volume of sludge needs to be managed and discarded properly. The most common method of treatment for these sludge is digestion . The lagooning, composting, and landfilling are the next methods of treatment. Mechanical dewatering may also be implemented as final treatment to reduce the volume of the stabilized sludge.Tajrishi  estimate5 and TSS parameters of 32.96 ± 1.91, 37.31 ± 2.44 g/d.cap obtained in this study are considerably lower than the values of 40 to 50 and 50 to 60 g/d.cap, respectively, recommended by Department of Energy of Iran for the design of new treatment plants. The per capita loading of wastewater were used by designers for the design of a wastewater treatment plant in Tehran, was estimated to be 215 L/d.cap while the actual per capita collected wastewater according to the real data of treatment plants was determined to be 186.06 ± 7.85 L/d.cap. This disparity has interesting implication when these estimated values are applied to the design of new wastewater treatment plant and calculation of an equivalent population for an industrial waste. The design of new treatment plant in Tehran with a per capita of 186.06 ± 7.85 L/d.cap may lower the required capacity for the treatment of wastewater up to 40%. The results of this study reveal that the Tehran’s wastewater may be classified as highly degradable, but during recent decades the Biodegradability Index has been reduced up to 15%. This may be resulted from this fact that over the past 15-20 years, a wide range of diverse industrial synthetic detergents have been produced and extensively used for cleaning and disinfection purposes and then discharged into sewage systems. According to the PDC of BOD5, Tehran was found to be with lower priority of BOD5 discharge reduction from domestic wastewater. It is estimated that 504 m3/d and 346 m3/d of sludge will be produced and waste as excrement raw sludge, respectively, in Tehran.In this study, the basic characteristic data regarding to the nine main domestic wastewater treatment plants in Tehran was assessed and analyzed. The BODIn conclusion, the use of new revised per capita parameters obtained in this study could help in designing more efficient treatment systems and generating more reliable data for operational control of wastewater treatment process in Tehran. However, further research on wastewater quality and quantity assessment and adequate monitoring measures are required for existing and future treatment facilities in Tehran to ensure that they comply with safe operational and environmental standards.aRegional Organization for the Protection of the Marine Environment.3/month)Nomin; the nominal capacity of WWTP (m3/month)Collect; the collected volume of wastewater (m3/month)Treat; the treated volume of wastewater (m3/month)ProSlu; the produced sludge (m3/month)ExcSlu; the excess sludge pHin; the Influent pH5 (mg/L)BODin; the Influent BODCODin; the influent COD (mg/L)TSin; the influent total solids (mg/L)TSSin; the influent suspended solids (mg/L)TDSin; the influent dissolved solids (mg/L)Pin; the influent phosphorous (mg/L)OrNin; the influent organic nitrogen (mg/L)TKNin; the influent total Kjeldahl nitrogen (mg/L)Tout; the effluent temperature (C°)pHout; the effluent pH5 (mg/L)BODout; the effluent BODCODout; the effluent COD (mg/L)TSout; the effluent total solids (mg/L)TSSout; the effluent suspended solids (mg/L)TDSout; the effluent dissolved solids (mg/L)Pout; the effluent phosphorous (mg/L)OrNout; the effluent organic nitrogen (mg/L)TKNout; the effluent total Kjeldahl nitrogen (mg/L)Slu_E; the generation per capita per day of excess sludge (L sudge/d.cap)GCPDSlu_P; the generation per capita per day of produced sludge (L sludge /d.cap)GCPDTS; the generation per capita per day of total solids (g /d.cap)GCPDOrgN; the generation per capita per day of organic nitrigen (g /d.cap)GCPDTDS; the generation per capita per day of total dissolved solids (g /d.cap)GCPDP; the generation per capita per day of total phosphorous (g /d.cap)GCPDTKN; the generation per capita per day of total kjeldahl nitrogen (g /d.cap)GCPDTSS; the generation per capita per day of total suspended solids (g /d.cap)GCPDCOD; the generation per capita per day of chemical oxygen demand (g /d.cap)GCPDBOD5; the generation per capita per day of biological oxygen demand (g /d.cap)GCPD5/COD)out; the biodegradability index of effluent wastewater(BOD5/COD)in; the biodegradability index of influent wastewaterTretPerP; the per capita per day of collected wastewater (L/d.cap)Coll


Expectant parents of very preterm infants, physicians, and policy makers require estimates for chances of survival and survival without morbidity. Such estimates should derive from a large, reliable, and contemporary data base of easily available items known at birth.To determine short-term outcome and risk factors in very-low-birth-weight preterm infants based on administrative data.Anonymized routine data sets transmitted from hospital administrations to statutory health insurance companies were used to assess survival and survival free of major morbidities in a large cohort of preterm infants in Germany.After exclusion of infants with lethal malformations, there were 13,147 infants with a birth weight below 1,500 g admitted to neonatal care 2008–2012, of whom 1,432 infants (10.9%) died within 180 days. Estimated 180 days survival probabilities were 0.632  for infants with 250–499 g birth weight, 0.817 (0.799–0.834) for 500–749 g, 0.931 (0.920–0.940) for 750–999 g, 0.973 (0.967–0.979) for 1,000–1,249 g, and 0.985 (0.981–0.988) for 1,250–1,499 g. Estimated probabilities for survival without major morbidity  were 0.433 (0.384–0.481) for 250–499 g, 0.622 (0.600–0.643) for 500–749 g, 0.836 (0.821–0.849) for 750–999 g, 0.938 (0.928–0.946) for 1,000–1,249 g, and 0.969 (0.964–0.974) for 1,250–1,499 g, respectively. Prediction of survival and survival without major morbidities was moderately improved by adding sex, small for gestational age, and severe or moderate congenital malformation, increasing receiver operating characteristic areas under the curve from 0.839 (0.827–0.850) to 0.862 (0.852–0.874)  and from 0.827 (0.822–0.842) to 0.852 (0.846–0.863) , respectively.The present analysis encourages attempts to use administrative data to investigate the association between risk factors and outcome in preterm infants. Kaplan–Meier analysis was used to describe survival up to 180 days and survival without major neonatal morbidity, with strata being compared by the log-rank test. The impact of predictor variables was assessed with adjusted odds ratios (OR) in multiple logistic regression analyses. To describe the power to predict survival and survival without major neonatal morbidity, receiver operating characteristics (ROC) curves were generated, and the areas under the curve with 95% confidence intervals were calculated.The study received institutional review board approval . No consent was required as this is a retrospective analysis of anonymized data collected according to federal law.n = 17; lethal malformations: n = 128, twins with identical numbers: n = 17). The characteristics of the 13,147 patients included are given in Table p = 0.0216) and small-for-gestational-age babies  in infants with a birth weight of 250–749 g, as compared to infants of 750–1,499 g, while the opposite was observed for multiples .A total of 13,303 infants covered by the regional statutory health insurance companies were born between January 1, 2008 and December 31, 2012 with a birth weight between 250 and 1,499 g. Of these, 156 were excluded  in the total group, and 22%  in infants below 1,000 g birth weight  infants. Estimated probabilities of 180 days survival without major morbidity were 0.433 (0.384–0.481) for 250–499 g, 0.622 (0.600–0.643) for 500–749 g, 0.836 (0.821–0.849) for 750–999 g, 0.938 (0.928–0.946) for 1,000–1,249 g, and 0.969 (0.964–0.974) for 1,250–1,499 g, respectively , sex, small for gestational age, multiple gestation, moderate, and severe congenital malformations, on survival and survival without major morbidity , and 0.827 (0.822–0.842) when only birth weight was used  and 0.852 (0.846-0.863) taking the 2–0.874 aThis analysis reports an overall 180 days survival rate of preterm infants with a birth weight <1,500 g admitted to neonatal intensive care units in Germany 2008–2012 of 89.1%, with 14.1% of deaths occurring between 30 and 180 days of life. Surgically treated major morbidities were observed in 7.7% of all very-low-birth-weight infants, and 7.8% of survivors, respectively. Rates of survival strongly declined alongside decreasing birth weight, from 97.9% in 1,250–1,499 g birth weight infants to 38% in infants below 500 g birth weight, while the rates of surgically treated major morbidities in survivors increased from 1.7 to 32%.We compared our data with published data of a cohort of 2,207 infants below 1,500 g birth weight without lethal malformations treated in 2010 in a network of 46 tertiary care neonatal units in Germany . The puba priori selected variables on death and death and major morbidity combined was remarkably similar. Multiple gestation was not linked to poor outcome, an observation made for recent cohorts also in Australia and New Zealand . This drawback is to be balanced against the risk of recruitment bias in prospective clinical trials, especially those that require antenatal parental consent , the risOur investigation was based on data from patients rather than cases, whereas events occurring after discharge or transfer to another hospital are being missed in unit-based network and quality improvement data sets. In contrast to other studies, the 180 days window allowed to take events after discharge or transfer to another hospital into account, unless there was a change in the company providing health care insurance. Late deaths are an important issue, especially in very small infants, with 13.3% of deaths occurring after 30 days of life in infants below 1,000 g birth weight. For comparison, 17.3 and 19.8% of all deaths occurred beyond 28 days of life in the American NICHD , respectAs a major weakness, some variables of great interest to clinicians are missing, particularly gestational age at birth, antenatal steroids, mode of delivery, and inborn/outborn status. A merger of the anonymized infant administrative data sets, as used in this study, with maternal records is being hampered by current rules governing privacy protection. The variable “small for gestational age” partly made up for the missing variable “gestational age at birth,” also avoiding the multicollinearity problem when birth weight and gestational age are entered simultaneously as continuous measures. The variable “antenatal steroids” has lost statistical impact as a result of the high rates of antenatal steroids and exogenous surfactant replacement therapy . As dataHigh-grade IVH, high-grade ROP, and NEC requiring surgery have been shown to be strong predictors of long-term neurodevelopmental impairment , 28–35. Data entered into any type of registry are liable to inadvertent mistakes, biased coding, or even wilful deceit. When estimating the risk of false entries, there are several questions to be addressed: (1) How reliable is the information given to the person who enters the data? (2) How likely are errors based on negligence? (3) Are there incentives for up-coding, down-coding, or omission of data? For these reasons, we chose to not include bronchopulmonary dysplasia as an outcome variable in this analysis, as it lacks an unambiguous marker to disassociate mild from severe forms unless strict definitions are being employed. Some degree of disordered lung development is common in very preterm infants, and it largely depends on the exact criteria used whether or not infants are classified as having bronchopulmonary dysplasia . For morAdministrative data have been used successfully for as diverse purposes as to estimate live delivery rates after tubal sterilization reversal , to compEJ, AB, CG, TB, GH, HH, and CB all contributed to the conception and design of this analysis. EJ, AB, and CG provided the statistical analysis, EJ and CB drafted the first manuscript that was revised by AB, CG, TB, GH, and HH and finally approved by all authors.The authors declare that this work was done in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.