Cytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. It can be difficult to differentiate with other diseases such as 21-hydroxylase deficiency (21-OHD), polycystic ovary syndrome (PCOS) and Antley–Bixler syndrome (ABS). Nearly 100 cases of PORD have been reported worldwide. However, the genetic characters and clinical management are still unclear, especially in China.POR gene (R457H) and her parents carrying the heterozygous mutation. The patient was treated with low-dose corticosteroids and estrogen/progesterone sequential therapy, and her ovarian cyst gradually reduced with regular menstruation in the follow-up. Moreover, the clinical and genetic characteristics of 104 previously reported PORD cases were also summarized and analyzed.In this study, we report a 27-year-old female Chinese patient who first presented with amenorrhea and recurrence of large ovary cyst. She was misdiagnosed with PCOS and non-classical 21-OHD due to ovary cysts and elevated 17-hydroxy-progesterone. The patient’s complaining of a mild difficulty of bending the metacarpophalangeal joints reminded us to consider PORD, which usually presents with skeletal deformities and sexual dysfunction. The diagnosis of PORD was confirmed by genetic analyses, which showed the patient harboring a homozygous missense mutation in the POR gene also give us a viewpoint that R457H mutation in POR gene maybe important in causing PORD in Chinese as same as in Japanese.PORD is a very rare disease which can be easily misdiagnosed in mild cases. Clinicians should keep in mind of this disease in patients with sexual dysfunction, especially combined with special skeletal deformities. Our data could provide a consciously understanding of this disease for clinic practicers. Low-dose corticosteroids combined with estrogen/progesterone sequential therapy will be effective in PORD patients with recurrence of large ovary cyst. The fact that the reported PORD patients in China carrying an identical variant R457H in  POR gene. POR transfers electrons from NADPH to cytochrome P450 enzymes for drug and toxin metabolism as well as steroid hormone synthesis  is encoded by the sis Fig.  1]. Sin. SinPOR nce 2004 . CurrentPORD can cause reduced activities of multiple enzymes participating in the synthesis of both adrenal and gonadal steroid hormones, which further leads to the dysfunction of the sex hormones and a decrease in glucocorticoids. Therefore, the disease is a type of special congenital adrenal hyperplasia (CAH). PORD is very rare and exhibits a variety of clinical manifestations, such as oligomenorrhea and ovarian cysts which resemble polycystic ovary syndrome (PCOS), elevated 17-hydroxy-progesterone (17OHP) which resembles 21-hydroxylase deficiency (21-OHD), and skeletal deformities which resemble Antley–Bixler syndrome (ABS). Therefore, in some cases, it can be difficult to differentiate PORD from PCOS, 21-OHD and ABS. However, PORD usually doesn’t exhibit hyperandrogenism which differs from PCOS and 21-OHD. In this study, we report a female adult Chinese PORD case who was misdiagnosed many times, and analyze the clinical presentation and genetic character of the patient and her parents.st affiliated hospital of China Medical University (Shenyang) for amenorrhea with an unknown etiology. The patient had labial fusion when she was born and underwent plastic surgery afterward. During puberty, the patient underwent another surgery for an ovarian cyst. The patient was treated in another hospital as PCOS for several times resulting with mild remission of amenorrhea and recurrence of ovarian cysts. The patient was suspected of having 21-OHD due to increased 17OHP levels and came to our hospital for a genetic test for CYP21A2 mutations. The patient did not have fatigue, loss of appetite or other symptoms and was the only child in the family. Her parents appeared to be normal. She was born by vaginal delivery, and the mother had no complaints of abnormal manifestations during the perinatal period or history of reproductive system diseases. Her parents are nonconsanguineous. Physical examination of the patient revealed the following characteristics: a height of 165 cm and weight of 55 kg; no hyperandrogenism symptoms like hirsutism or acne; and no purple stripes. The patient complained of difficulty of bending the metacarpophalangeal joints from childhood  patient with normal cognition visited the Endocrinology Clinic of 1ood Fig. . There wThe ultrasound showed the presence of a 1.2 × 1.4 cm fluid area in the left ovary, and there was a 9.5 × 6.3 × 4.3 cm cyst in the right ovary behind the uterus. Digital radiography (DR) suggested there were no bone abnormalities Fig. . The adrCYP21A2 (NM_000500) was tested first due to the suspicion of 21-OHD, but no mutations were found in the patient. Then the patient’s complaint about a mild difficulty of bending the metacarpophalangeal joints reminded us to consider PORD, which usually presents with skeletal deformities and sexual dysfunction, so POR (NM_000941) gene was tested secondly. Exon 11 of PORharbored a homozygous mutation (c.1370G > A) which leads to a conversion of arginine at amino acid position 457 to histidine (R457H). The patient’s parents were both heterozygous carriers of this variant  and estrogen/progesterone sequential therapy in our hospital, and her ovarian cysts gradually decreased in size (the biggest ovarian cyst shrank to 3.5 × 3.3 × 2.3 cm) with regular menstruation in the following visits. The serum levels of ACTH, LH, 17OHP and P were declined accordingly  was reported; however, no mutations were found in the patient in  CYP19A1 . POR donMost of PORD patients presented with ambiguous genitalia (75%). The pathogenic mechanisms of this manifestation are associated with impaired syntheses of testosterone, dihydrotestosterone and conversions of the androgens to estrogens. These syntheses and conversions depend on CYP17A1, CYP19A1, CYP21A2, which require POR , 9. In aSimilar to 21-OHD, despite that the basal levels of ACTH and cortisol are generally normal in PORD patients, the ACTH stimulation test showed that most of the patients (74.6%) have insufficient adrenocortical reserve. Activities of different POR mutants for target enzymes would have a certain role in variability of adrenocortical insufficiency –13. PatiPOR lead to the reduced activity of CYP51A1, thus reducing the synthesis of meiosis-activating sterol and further inducing a dysfunction in meiosis and the maturation of the oocytes. Under the above-mentioned “double hit”, compared with ovarian cysts associated with other hormone synthesis disorders, those in PORD are more difficult to control. Surgical treatment combined with corticosteroid and sex hormone replacement therapy is often required to prevent recurrence [Ovarian cysts were found in this patient and in other adult female patients (46.7%). If patients have a history of amenorrhea or anovulation, the disease is easily misdiagnosed as PCOS at primary medical units. The decrease in estrogen causes hypergonadotropic hypogonadism, which stimulates the growth of the ovaries. On the other hand, mutations in currence . Our patPOR gene are associated with more serious skeletal deformities. This may also result from the effect of various mutations on the activity of related enzymes [Another important clinical feature of PORD is that there are different degrees of Antley-Bixler syndrome-like skeletal deformities (82.7%). Skeletal deformities include midface hypoplasia, craniosynostosis, foot and hand deformity, bony union of the large joints and femur bending. Some researchers have attempted to quantify these skeletal deformities and assess the severity according to quantitative indices. They found that compound heterozygous and homozygous mutations in the  enzymes . Our pat enzymes  and reti enzymes .P450 liver enzymes and heme oxygenase involving in drug metabolism need POR to provide electrons, therefore, the mutation of POR gene or associated SNPs may affect drug metabolism in human body , 20 and POR mutations and single nucleotide polymorphisms (SNPs) [http://www.cypalleles.ki.se/por.htm). According to the cases reported in the literature, the most common mutation type in the Japanese population is R457H , and it is A287P  in Europeans [It has been reported that there are nearly 200 s (SNPs) , includiH 51/78, 5.4%, anduropeans , 25–33, uropeans , but thiuropeans . There aPOR gene also give us a viewpoint that R457H mutation in POR gene maybe also important in causing PORD in Chinese, which is similar with Japanese.In conclusion, PORD is a very rare disease which can be easily misdiagnosed in mild cases. Clinicians should keep in mind of this disease in patients with sexual dysfunction, especially combined with special skeletal deformities. Our data could provide a consciously understanding of this disease for clinic practicers. Low-dose corticosteroids combined with estrogen/progesterone sequential therapy will be effective in PORD patients with recurrence of large ovary cyst. The fact that the reported PORD patients in China carrying an identical variant R457H in


By 2050, nearly 70% of the global population is projected to live in urban areas. Because the environments we inhabit affect our health, urban and transport designs that promote healthy living are needed.We estimated the number of premature deaths preventable under compliance with international exposure recommendations for physical activity (PA), air pollution, noise, heat, and access to green spaces.We developed and applied the Urban and TranspOrt Planning Health Impact Assessment (UTOPHIA) tool to Barcelona, Spain. Exposure estimates and mortality data were available for 1,357,361 residents. We compared recommended with current exposure levels. We quantified the associations between exposures and mortality and calculated population attributable fractions to estimate the number of premature deaths preventable. We also modeled life-expectancy and economic impacts.We estimated that annually, nearly 20% of mortality could be prevented if international recommendations for performance of PA; exposure to air pollution, noise, and heat; and access to green space were followed. Estimations showed that the greatest portion of preventable deaths was attributable to increases in PA, followed by reductions of exposure to air pollution, traffic noise, and heat. Access to green spaces had smaller effects on mortality. Compliance was estimated to increase the average life expectancy by 360  days and result in economic savings of 9.3  billion EUR/year.PA factors and environmental exposures can be modified by changes in urban and transport planning. We emphasize the need for a) the reduction of motorized traffic through the promotion of active and public transport and b) the provision of green infrastructure, both of which are suggested to provide opportunities for PA and for mitigation of air pollution, noise, and heat.Mueller N, Rojas-Rueda D, Basagaña X, Cirach M, Cole-Hunter T, Dadvand P, Donaire-Gonzalez D, Foraster M, Gascon M, Martinez D, Tonne C, Triguero-Mas M, Valentín A, Nieuwenhuijsen M. 2017. Urban and transport planning related exposures and mortality: a health impact assessment for cities. Environ Health Perspect 125:89–96; http://dx.doi.org/10.1289/EHP220 By 2050, nearly 70% of the global population is projected to live in urban environments . Cities Physical inactivity and ambient air pollution are estimated to cause more than five million premature deaths each year worldwide and rank among the leading risk factors in the Global Burden of Disease Study . Car-cenIn addition to being the main source of air pollution in urban areas, motorized road traffic exposes an estimated 40% of Europeans to daytime noise levels exceeding the WHO recommended threshold of 55 dB ; it alsoWe aimed to estimate the mortality burden associated with exposures related to current urban and transport planning. For this purpose, we developed the Urban and TranspOrt Planning Health Impact Assessment (UTOPHIA) model and conducted a health impact assessment (HIA) for Barcelona, Spain. We estimated the impact of meeting the international recommendations for performance of PA; exposure to air pollution, noise and heat; and access to green spaces on preventable natural all-cause mortality, life expectancy, and economic savings.2  using data from 2012. The analysis estimated the impact on natural all-cause mortality for Barcelona residents ≥ 20 years of age  under compliance with international exposure level recommendations. The 2012 natural all-cause mortality rate for Barcelona residents ≥ 20 years of age was 1,108 deaths/100,000 persons after excluding external causes of death (see Tables S1 and S2)  .a) We obtained recommended exposure levels ; and b) current exposure levels; c) we determined the difference between recommended and current exposure levels (“exposure difference”); d) we obtained the exposure response functions (ERFs) quantifying the association between exposure and mortality from the literature (e) we calculated the relative risk (RR) and (f) the population attributable fraction (PAF) for each exposure difference .We developed the UTOPHIA tool following quantitative HIA methodology  WHO and as folloterature ; (e) we Life expectancy and economic evaluations were also performed. We estimated average change in life expectancy based on age-specific all-cause mortality rates for Barcelona (2011)  followinPhysical activity. The WHO recommends that adults ≥ 18 years of age should achieve 150 min of moderate-intensity aerobic PA or 75 min of vigorous-intensity aerobic PA weekly (A weekly  (WHO 201A weekly .Air pollution. Particulate matter with a diameter ≤ 2.5 μm (PM2.5) is a commonly used proxy for exposure to all fossil fuel combustion sources (2.5 exposure concentrations should not exceed 10 μg/m3 ( sources . The WHO10 μg/m3 .Noise. The WHO recommends that daytime (0700–2300 hours) outdoor noise levels should not exceed equivalent sound pressure levels above 55 A-weighted decibels [dB(A)] ( [dB(A)] .Heat. Although there are no guidelines, increasing greenery and urban albedo while reducing traffic and impermeable surfaces in cities may provide cooling in the summer months by ≤ 4°C  through the 2011 Barcelona Health Survey, a population-based randomized sample studying the health status of Barcelona residents  minutes per week, respectively (see Tables S3 and S4) . The assa) a linear ERF and b) including METs accumulated by walking as part of total PA were performed .Sensitivity analyses using Air pollution. Annual mean PM2.5 data for 2012 were available for Barcelona at the census-tract level through the European Study of Cohorts for Air Pollution Effects Land Use Regression (ESCAPE LUR) model (2.5 concentrations necessary to comply with the recommendation of 10 μg/m3 was estimated for each census tract.R) model . The exp2.5 and mortality was quantified using a linear ERF (The association between PMnear ERF . The RR a) the WHO interim target of an annual mean of 15 μg/m3 for PM2.5 (b) the lowest measured PM2.5 level of 5.8 μg/m3 were performed   tand S10) .Noise. Daytime traffic noise levels were calculated at the census-tract level using Barcelona’s strategic noise map   . The ERFeq,16hr) , assuminAeq,16hr 55 dB(A). The corresponding RR and PAF were calculated based on the predicted ERF.The exposure difference was determined for each census tract exceeding LAs a sensitivity analysis, the PAF was calculated exclusively for the proportion of people in each census tract that self-reported noise annoyance  .Heat. Daily mean temperatures (2009–2014) were available through a central monitor in Barcelona  . BetweenFor days exceeding 21.8°C, the exposure difference in daily mean temperature was calculated at the census-tract level. The corresponding RR and PAF were calculated. Temperatures were theoretically reduced by 4°C, and the exposure difference was calculated for days still exceeding 21.8°C. The corresponding RR and PAF were calculated. The number of deaths attributable to temperatures reduced by 4°C was subtracted from the number of deaths attributable to the estimated temperatures for 2011.A sensitivity analysis with a temperature reduction of 1°C was performed .Green space. To provide universal access to a green space ≥ 0.5 ha within a 300-m linear distance, we estimated how much green space surface (%GS) each census tract needed to have.n = 3,417), the proportion of Health Survey respondents living within 300 m of a green space ≥ 0.5 ha was determined for each %GS quintile . A logarithmic function was fitted to predict the %GS needed to provide universal access to a green space ≥ 0.5 ha within 300 m . It was predicted that each census tract would need to have 25.6%GS in order to provide universal access to a green space ≥ 0.5 ha within 300 m.Green space data were available through Urban Atlas   and the The exposure difference between the current %GS of each quintile and the necessary 25.6% was determined . A linear ERF was used to quantify the association between green space and mortality . For eacMore than 70% of adults in Barcelona were insufficiently active . Air polAnnually, 2,904  deaths were estimated to be preventable if Barcelona complied with international exposure recommendations . EstimatUnder compliance with international exposure recommendations, Barcelona’s residents were estimated to live an average of 360  days longer, and an estimated 9.3  billion EUR could be saved annually.The results from the sensitivity analyses are presented in the Supplemental Material , and they indicate that our estimates are generally robust.We developed and implemented the UTOPHIA model for Barcelona and estimated that 2,904  of all annual natural deaths in Barcelona could be prevented if international recommendations for performance of PA; exposure to air pollution, noise, and heat; and access to green space were followed. The present study is the first to quantify the effects of multiple urban and transport planning–related exposures in a city, and we showed considerable impacts of these exposures on health.2.5 reductions with implementation of proposed transport policy measures would result in a reduction of premature mortality by 3%  . Other Hty by 3% .We have estimated that Barcelona’s compliance with international exposure recommendations would have a considerable impact on all-cause mortality. However, HIAs involve multiple assumptions that carry uncertainties in estimating health impacts and of which we could quantify only a limited extent.The ERFs for PA, air pollution, and green spaces were obtained from the most recent meta-analyses. The evidence for the mortality effects of PA and for air pollution is stronger than for the other exposures simply because more research has been done on these exposures. The estimates of noise and green spaces are only suggestive, as indicated by the wide confidence intervals. Despite emerging evidence on green spaces providing general health benefits , until nGenerally, benefit estimations are sensitive to the contextual setting and underlying population parameters. Estimations of health impacts depend largely on baseline exposure to the health pathways considered and the general health status of the population; thus, varying results can be expected in different settings. Moreover, personal choices and intrinsic motivations for behavior change , and thus exposure alterations, are unquantifiable, but they can have a large impact on health. Thus, the generalizability and causal inference of our results may be uncertain.In addition, time lags in benefit estimations and the resulting delayed receipt of health benefits can significantly alter benefit estimations. Because we were interested in long-term effects of exposure alterations, a delay in the receipt of benefits is expected. Practical implications of this delay may be that changes to urban and transport planning practices are less relevant for younger people in terms of mortality impacts, but its importance is reinforced for older people. In times of demographic change and increasing aging populations, it is important to keep this delay in mind. In this regard, the economic impact is most likely overestimated; here, time discounting applies because benefits occurring in the future are less valuable than benefits occurring immediately.2.5 mortality effects. Other traffic-related air pollutants , which we did not consider, have been shown to have independent mortality associations  and a common mitigator . Estimated effects might interact and synergies may exist between the exposures. At the present time, evidence of the independence of mortality effects is only available for air pollution and noise . Therefociations .The strength of this study is its novelty in terms of linking urban and transport planning–related exposures and health in an integrated way, which highlights the considerable impacts on mortality of noncompliant exposure levels. The detailed exposure data on the same spatial scale strengthen the internal validity of the study. The census-tract level and exposure models were of fairly refined resolution. The sensitivity analyses showed that our estimates were fairly robust.Despite being unable to show the extent to which improvements of the urban environment could actually contribute towards achieving recommended exposure levels, it is believed that reconsideration of urban and transport policies would have a large impact on PA factors and environmental exposures. Therefore, HIA is a valuable tool to enhance understanding of the interrelationship between the environment and health and can assist policy makers in optimizing health gains.n = 30 for 2012)  , trafficor 2012) .A paradigm shift in urban and transport planning is needed to provide a multidimensional approach to urban environmental quality and associated public health benefits . IncreasPhysical activity. Insufficient PA was associated with the greatest excess mortality in Barcelona; this highlights the urgency of integrating PA into daily life. Active and public transport provide a great opportunity to do so because both forms of transport provide coincidental health gains by increases in PA. Public transport is estimated to provide an additional 10 min of walking per day  , but furReinforcement of green infrastructure may also facilitate PA engagement  because exercise in green spaces is associated with higher-intensity exercising and increased enjoyment .Air pollution, noise, and heat. Exposure to air pollution, noise, and heat resulted in large contributions to the estimated mortality burden. Barcelona’s vehicle fleet of > 500,000 cars and nearly 300,000 scooters and motorcycles, and an additional daily suburban commuter fleet, result in a high volume of motorized traffic and associated emissions  and the provision of urban greening. Taking opportunities with urban renewal, densely constructed gray infrastructure could be loosened up and replaced with nonradiating and green infrastructure. Vegetation can be a passive control of air pollution exposure , is a naGreen space. Despite the suggested minor impact of green spaces on natural all-cause mortality, the co-benefits of PA engagement and refuge from harmful environmental exposures  make green spaces an important urban and traffic management tool.The present study evaluated mortality effects of access to green spaces. The recommendation of a 300-m linear distance is supported by research findings suggesting that green space use declines beyond 300–400 m . For acta) mitigation  (b) association of visual access to green spaces with stress relief, positive affect, and restoration (c) improved mental health (d) enriched biodiversity strengthening immune function (e) increased safety perception and social cohesion  ; b) assotoration ; c) imprl health ; d) enrifunction ; and e) cohesion .a) the reduction of motorized traffic through promotion of active and public transport and b) the provision of urban greening; both of these factors can provide opportunities for PA engagement as well as mitigation of air pollution, noise, and heat.Each year in Barcelona, nearly 20% of mortality was estimated to be attributable to noncompliance with recommended levels of PA, air pollution, noise, heat, and access to green spaces. Environmental exposures and PA factors can be modified by changes in urban and transport planning. We urge the consideration of health impacts when designing cities and emphasize the importance of (522 KB) PDFClick here for additional data file.


Opisthomonorchis and Paramonorcheides, have been investigated, for the first time, by means of transmission electron microscopy. The ultrastructural study reveals that the mature spermatozoon of Opisthomonorchis dinema and Paramonorcheides selaris share several characters such as the presence of two axonemes of different lengths showing the 9+“1” pattern of the Trepaxonemata, a nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies, granules of glycogen and similar morphologies of the anterior and posterior extremities. The slight differences between the male gamete of O. dinema and P. selaris are the length of the first axoneme and the position of the second mitochondrion. This study also elucidates the general morphology of the spermatozoon in all monorchiid species described so far, which corresponds to a unique spermatozoon type. Other interesting finds concern the spermatological similarities between monorchiid spermatozoa and the mature spermatozoon reported in the apocreadiid Neoapocreadium chabaudi. These similarities allow us to suggest a close phylogenetical relationship between the Monorchiidae and the Apocreadiidae, although more studies are needed, especially in the unexplored taxa.Spermatological characteristics of species from two monorchiid genera,  Monorchis parvus and Helicometroides atlanticus  caught off Nouméa, New Caledonia, from the same fish specimen (MNHN JNC3224) as the paratypes of the species   caught off Nouméa, New Caledonia, on 10 Sept. 2009 (fish specimen MNHN JNC 3043).Fish were bought dead, but very fresh, at the fishmarket in Nouméa, New Caledonia. Specimens of  species . Specime®). The sections were placed on 300 and 200 mesh copper grids and stained with uranyl acetate and lead citrate according to Digenean specimens were fixed in cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2, rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, post-fixed in cold (4°C) 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and propylene oxide series, embedded in Spurr resin and polymerized at 60°C for 24 h. Ultrathin sections (60–90 nm) of the seminal vesicle were obtained on an ultramicrotome .Opisthomonorchis dinema and Paramonorcheides selaris is described after observations of cross- and longitudinal sections .The organization of the mature spermatozoon of sections –4. Thus,O. dinema the anterior spermatozoon tip exhibits both anterior extremities of the axonemes, surrounded by a layer of cortical microtubules (about 26) and external ornamentation  are also needed to test their close relationships with the Monorchiidae.Opisthomonorchis dinema and Paramonorcheides selaris share several ultrastructural features with those reported in Monorchis parvus and Helicometroides atlanticus (The mature spermatozoa of lanticus . These s –Presence of an anterior extremity showing two axonemes accompanied by cortical microtubules and external ornamentation, –Presence of the association “external ornamentation+cortical microtubules”, –Location of the external ornamentation corresponding to the type 1 according  –Presence of two bundles of parallel cortical microtubules and two mitochondria, –Posterior spermatozoon extremity containing only the nucleus according to Neoapocreadium chabaudi (The model of mature spermatozoon described here for the monorchiid species is relatively similar to that reported to the apocreadiid chabaudi . These sThe ultrastructural characteristics described in digenean and in particular, the Monorchiidae could be used as phylogenetic tools and when establishing spermatozoon models, given that they may allow the distinguishing of genera, families or superfamilies within the Digenea.


Volume reduction of the pancreatic tissues following a pancreatectomy can lead to the deterioration of glucose homeostasis. This is defined as pancreatogenic diabetes mellitus (DM). The objective of this study was to investigate the occurrence of new-onset DM (NODM) and evaluate the risk factors, including the pancreas volume reduction rate in patients undergoing pancreaticoduodenectomy (PD).Sixty-six patients without preoperative DM underwent PD for periampullary tumors between August 2007 and December 2012 and were included in this analysis. These patients underwent follow-up tests and abdominal computed tomography (CT) scan 7 days, 6 months, 12 months, 24 months, and 36 months after the operation. The pancreas volume reduction rate was calculated by CT volumetry. The patients were divided into 2 groups according to the postoperative development of DM.P = 0.002).After PD, newly diagnosed DM occurred in 16 patients (24.2%). The incidence of DM was highest among patients with carcinomas with an advanced T stage. The pancreatic volume reduction rate after 6 and 12 months in the NODM group was significantly higher than the normal glucose group in the univariate analysis. In the multivariate analysis, the pancreatic volume reduction rate 6 months after PD was the only significant predictive factor for the development of NODM (This study suggests that the pancreatic volume reduction rate 6 months after PD was the only significant predictive factor for the development of NODM. CT volumetry of the pancreas may be useful as a predictor of NODM after PD. Recent development of better diagnostic modalities and a better understanding of pancreatic disease has increased the frequency of pancreatectomies in benign or premalignant pancreatic disease. Distal pancreatectomy (DP) has been successful for various pancreatic diseases, leading to long-term survival. Because of the success in mortality rates, several studies have now focused on the morbidity of the surgery, namely, new onset DM. Several research studies have reported that new onset DM developed in 4.8% to 38% of patients after DP.,6–8 And, after PD, the incidence of new onset DM was reported between 0% and 50%.,6–16 However, with pancreaticoduodenectomy (PD), lower survival rates and poor prognosis have been reported due to the high rate of pancreatic head cancer compared to other malignant tumors in the periampullary lesion. For this reason, new onset DM after PD has received very little attention. However, there is a concern that underestimation of the incidence and clinical importance for new onset DM might cause decreased quality of life by delaying proper medical therapy. Additionally, it is important to quantify the risk of new onset DM because other malignancies and premalignant lesions for which PD is performed, such as ampulla of Vater cancer or intraductal papillary mucinous neoplasm, have expectation for long-term survival. Although there is more research on DP than for PD in this matter, reliable risk factors for new onset DM have not been determined due to limitations of the studies. Recently, some studies attempted to predict new onset DM by assessing the degree of pancreatic volume reduction using CT volumetry. The objective of this study was to investigate the frequency and risk factors of new onset DM in patients who have undergone PD and had long-term survival. In addition, we performed pancreatic volume analysis to examine the relationship between the serial reduction in pancreatic volume and the development of new onset DM, perhaps revealing the importance of evaluating glucose metabolism during follow-up.Pancreatic resection could be a risk factor for new onset diabetes mellitus (DM) due to the deterioration of glucose metabolism caused by volume reduction of endocrine and exocrine pancreatic tissues.22.1We analyzed 113 patients who underwent PD for periampullary tumors at Pusan National University Hospital between August 2007 and December 2012. We excluded patients with preoperative DM or who had chronic pancreatitis or those who were lost to follow-up or who survived less than 3 years. Sixty-six patients met the criteria and were enrolled in the study. The patients were divided into 2 groups according to the postoperative development of DM: non-DM group (n = 50) and postoperative DM group (n = 16). These patients were retrospectively reviewed for age, sex, body mass index (BMI), postoperative pancreatic fistula (POPF), postoperative adjuvant therapy, pathologic characteristics, presence of cardiovascular comorbidity and operative factors  based on the medical records. These patients also had the volume of their pancreas measured by computed tomography (CT) volumetry before surgery, and then the remnant was measured on postoperative day 7 and months 6, 12, 24 and 36. This retrospective study was approved by the institutional review board at Pusan National University Hospital Clinical Trial Center (IRB No. E2016047), and written informed consent was obtained from all participants.2.2Dynamic CT was performed using a 64-MDCT scanner . Images of the arterial, portal venous, and delayed phases were obtained using a helical scanning technique. The parameter of the CT scanners was 120 kVp and 0.625 mm collimation with a pitch of 0.984. Automatic tube current modulation was applied with a setting from 180 to 350 mA. One hundred twenty milliliters of Omnipaque 300  was injected at a rate of 3 mL/s. Arterial phase, pancreatic phase and delayed phase imaging were acquired at 35, 70, and 125 seconds, respectively, after the contrast material was injected. The axial images were reconstructed at a 3-mm section thickness. Three-dimensional (3D) images were reconstructed by TeraRecon Aquarius Workstation v4.4  using an LD2 algorithm  with axial images , fasting plasma glucose of ≥126 mg/dL after fasting for ≥8 hours, or a 2-hour postprandial glucose level more than 200 mg/dL at follow-up.2.4t test or the Mann–Whitney U test, where appropriate. A logistic regression model was used for univariate and multivariate analyses. The cutoff value of the pancreas volume reduction rate for predicting new onset DM was determined by a receiver operating characteristic (ROC) curve analyses. P values less than 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS version 23.0 for Windows .Data distribution was verified by the Shapiro–Wilk test. Categorical variables between 2 groups were compared using the Chi-square or Fisher exact test. The continuous variables were compared using Students 32. Pylorus preserving pancreaticoduodenectomy (PPPD) was performed in 44 patients (66.7%), and 22 patients (33.3%) underwent PD. The tumor locations included the pancreatic head , the bile duct , ampulla of Vater , and the duodenum . Postoperative pathologic results revealed malignancy in 60 patients, while the pathology was benign in the other. The benign diseases included intraductal papillary mucinous neoplasm, solid and papillary epithelial neoplasm, and paraduodenal pancreatitis. The median duration of follow-up after surgery was 40 months. Postoperative new-onset DM (NODM) had developed in 16 (24.2%) of the 66 patients at an average of 14.8 months after surgery.A total of 66 patients were included in the analysis. Demographics and clinical characteristics of the patients are summarized in Table 2, operation type, transfusion, pancreatic fistula, adjuvant treatment, N stage, recurrence, tumor size, cardiovascular comorbidity, cholesterol level, anesthesia time, perineural invasion, and lymphvascular invasion were not significantly different between the normal and new onset DM groups. An advanced T stage was identified as a risk factor for new onset DM after PD in univariate analysis (P = 0.035).Comparison of clinicopathologic factors between 2 groups are described in Table P = 0.097). The pancreatic volume reduction rate after 6 and 12 months in new onset DM group was significantly higher than in the normal glucose group  (Table P = 0.002) (Table P = 0.000) .The mean pancreatic resection volume on CT volumetry was 36.60 ± 18.13 mL. The mean pancreatic resection volume did not differ between the normal glucose group and the new onset DM group  and new onset DM and have shown that the pancreatic volume status after surgery did not significantly predict new onset DM.,15There has been very little research conducted examining the relationship between pancreatic volume status after pancreatectomy, especially PD and new onset DM. Thus, the association between the volume status after pancreatectomy and new onset DM is unclear and controversial. The exact volume of pancreas or resection volume cannot readily be determined during surgery. Therefore, the volume of the remnant pancreas after PD can be indirectly measured by CT volumetry.In the present study, our analysis also demonstrated that the resection volume during PD was not associated with new onset DM. However, our multivariate logistic regression analysis indicated that the pancreatic volume reduction rate 6 months after PD independently predicted the risk of new onset DM. This result indicated that atrophy of the remnant pancreas might be a more significant risk factor for new onset DM than the initial resection volume of the pancreas. The pancreatic volume reduction rate at 6 and 12 months after PD in the new onset DM group was significantly higher than the normal glucose group in the univariate analysis. This result proposes that the remnant pancreas volume decreased gradually until 12 months after PD. Therefore, postoperative atrophy of the remaining pancreas may significantly increase the risks of new onset DM. Although the mechanism of postoperative atrophy is not well clarified, a suggested mechanism involved the loss of the gastrointestinal hormones, such as cholecystokinin and gastrin, due to resection of the duodenum and distal stomach. They described that the mean time interval between pancreatic resection and onset of DM was 16.8 months and suggested that careful analysis of glucose metabolism parameters is necessary, especially at approximately 16 months after pancreatic resection.In the 16 patients who developed new onset DM, the mean interval between PD and new onset DM was 14 months. This finding may be associated with our results, in which the pancreatic volume reduction at 6 and 12 months after PD is related to new onset DM. Similar results have been reported by Kwon et al.The difference between our research and previous studies is that by serial measurement of the remnant pancreatic volume, we drew the conclusion that the volume decrease at a particular point was associated with new onset DM. Based on our results and previous studies, the regular assessment of DM and follow-up should be considered within 18 months. The degree of atrophy appears to become stable within 18 months after surgery, thus, the rate of new onset DM is likely to decrease after that time point. In addition, this study indicates that if the pancreatic volume reduction rate is greater than 64% by 6 months, new onset DM can be predicted with a sensitivity of 75% and a specificity of 78% (AUC = 0.809). Therefore, if more than a 64% reduction is observed on CT volumetry, an evaluation for new onset DM should be performed.The limitations of this study include its retrospective nature and the limited number of patients. We did not address why some patients developed more losses of pancreatic volume due to loss of the gastrointestinal hormones. And, the relationship between stricture of pancreaticojejunostomy and NODM in patients developing DM is not well clarified in this study. Additional large studies will be needed to clarify the predictive risk factors for new onset DM and the effect of pancreatic atrophy on new onset DM after PD.5In conclusion, after PD, postoperative NODM had developed in 24.2% of the nondiabetic patients. The pancreatic volume reduction rate after 6 months after PD was the only significant predictive risk factor for the development of new onset DM. The remnant pancreas volume decreased gradually until 12 months after PD. Therefore, postoperative atrophy of the remaining pancreas may significantly increase the risks of new onset DM. In addition, patients who undergo PD should be evaluated for new onset DM in order to improve their quality of life after surgery. Pancreatic volumetry may be a useful predictor for new onset DM during follow up.


Porphyra, red algae) where the gametophyte is the dominant phase in the life history. Three Porphyra species  were used in comparisons of ploidy levels, genome sizes and genetic differentiation using flow cytometry and 11 microsatellite markers among putative polyploid lineages. Multiple ploidy levels and genome sizes were found in Porphyra species, representing different cell lines and comprising several cytotype combinations among the same and different individuals. In P. linearis, genetic differentiation was found among three polyploid lineages: triploid, tetraploid and mixoploids, representing different evolutionary units. We conclude that the gametophytic phase (n) in Porphyra species is not haploid, contradicting previous theories. New hypotheses for the life histories of Porphyra species are discussed.Whole genome duplication is now accepted as an important evolutionary force, but the genetic factors and the life history implications affecting the existence and abundance of polyploid lineages within species are still poorly known. Polyploidy has been mainly studied in plant model species in which the sporophyte is the dominant phase in their life history. In this study, we address such questions in a novel system ( In angiosperms, many species have been suggested to have polyploid ancestry2. While whole genome duplication (WGD) is now accepted as an important evolutionary force, the genetic factors and the life history implications affecting the existence and abundance of polyploid lineages are still poorly understood5. Recent studies have also demonstrated that polyploid genomes can be highly dynamic and undergo rapid structural and functional changes. These findings have renewed the interest in examining WGD as an evolutionary process7, which coupled with molecular genetics and phylogenetic analyses can provide new insights into the evolution of the genome8.Polyploidy, the increase in genome size by the acquisition of more than one set of chromosomes has been a key factor in eukaryote evolution. In fact, most flowering plants and vertebrates descend from polyploid ancestorsBrassica, Cotton, Maize, Spartina, Wheat, Tragopogon) representing life cycles in which the sporophyte is the dominant phase in a complex cycle. This cycle alternates between sporophytes (spore-producing organisms) and gametophytes (gamete-producing organisms)9. Although the sporophyte generation dominates in most extant lineages of land plants, in the earliest groups of land plants and also in several macroalgal groups, the gametophyte generation was (and it still is) the dominant component of the life cycle. Up to date, little is known about polyploidy in system groups that have the gametophyte as the dominant phase. This study uses red algae, an Archaeplastida group on the evolutionary line leading to plants, to study polyploid processes in a “gametophyte dominant” system. By studying polyploidy in such a life cycle, it may be possible to gain insights into polyploid evolution and to address questions related to the origins of polyploid lineages/individuals, the genetic consequences of genome duplication, and/or the importance of the triploid bridge in the origin of tetraploid populations, a topic still under debate12.In polyploid research, most of the systems studied are higher plants  have revealed more species diversity in Porphyra than was previously understood, little is known about the mechanisms of speciation in the genus Porphyra. Three of the recognized Porphyra species are the subject of this study: Porphyra dioica J Brodie & L. M. Irvine, Porphyra linearis Greville, and Porphyra umbilicalis Kützing. It is worth to note that although the gametophyte is the dominant phase in their life history, each species has a different mating system in Southern Europe: Porphyra dioica has mostly individuals with separate sexes (dioecious), P. umbilicalis can be hermaphroditic with both sexes in the same individual and gametangia separated in different sections of the thallus (monoecious) or also dioecious, and P. linearis has individuals with both systems, dioecious and hermaphroditic, but probably as a consequence of a protandrous system .Porphyra species typically have a heteromorphic life cycle consisting of a gametophytic blade phase (haploid) and a filamentous sporophytic phase called the conchocelis (diploid). The conchocelis phase of Porphyra species is difficult to find in nature18 with only a few in situ records (e.g. refs20), in contrast with the gametophytic blades, which are common and abundant in nature. The first description of the Porphyra life cycle by21 revolutionized the farming of this edible seaweed. Drew proved that Conchocelis rosea Batters, originally described as a separate species, was the germinated carpospores  from the Porphyra blades . It has been assumed since then than two ploidy levels are involved in the Porphyra life cycles and several chromosome studies have corroborated the ploidy level of both phases, with the gametophytic blade being haploid and the sporophytic carpospores/conchocelis being diploid (e.g. refs 25). Drew’s study enabled mass cultivation and the development of the aquaculture industry of Porphyra , the single most valuable marine product in the orient with a current retail value over $1.3 billion per year26.Porphyra species that are the subject of this study, to describe the population structure and historical biogeography of these species in the North Atlantic. These markers have allowed us to detect gametophytic blades of Porphyra possessing heterozygous genotypes with 1–6 alleles per individual in many samples along European coasts . These findings led us to question if different ploidy levels exist on different individuals/strains and or populations and whether these may be the result of hybridization with other Porphyra species/lineages. However, if polyploids exist in our populations/species, how these results can be integrated into the haploid/diploid life history assumed by21 is an enigma to be resolved. The origins of polyploid lineages, strains, or individuals within the species-complex, have yet to be determined.Recently, within the context of the project NORIGENOMICS (PTDC/MAR/099698/2008), microsatellite markers have been developed for the three Porphyra with different mating systems and, if present, to get insights into the mode of origin of polyploids . A second objective was to use the evidence from ploidy levels together with the estimation of genetic differentiation among the ploidy types found to understand if the species/cytotypes maintain gene flow. Finally, we expected that these results would provide insight into the evolutionary consequences of ploidy variation for the life history of species of Porphyra representing different mating systems; and to contrast hypothetical models for the evolution of polyploidy in organisms where the gametophyte is the dominant phase, relative to those where it is the sporophyte. Besides improving the understanding of the evolutionary dynamics in polyploid complexes in non-specialized plant species, our study provides evidence of a new evolutionary mechanism in the economically important genus Porphyra and that might perhaps be common in other red algae.The main objective of this study was to assess the existence of putative polyploid lineages in species of P. dioica, 40 blades; P. umbilicalis, 35 blades; and P. linearis, 47 blades) were collected in order to assess their ploidy levels, genome sizes and genetic differentiation among putative polyploid lineages. The localities visited were: Buarcos, Figueira da Foz   and Belém, Lisboa  (for P. linearis), both in Portugal. Each individual was examined in the binocular microscope and up to 4 sections were made per blade in duplicate  for further analyses: vegetative blade, zygotospores, female gametes and spermatia . The terms zygotosporangia and zygotospores are used to replace carposporangia and carpospores as proposed by Guiry27 for the Bangiales.A total of 122 blades  in 1 mL of WPB buffer28 to ensure that nuclei of both species were exposed to identical chemical and mechanical conditions. To double check for the existence of smaller genome sizes we also applied to a small set of samples a standard of smaller genome size, Raphanus sativus (2 C = 1.11 pg)29. The nuclear suspension was then filtered through a 30 μm nylon filter to remove large fragments, and 50 μg/mL of PI - Propidium iodide  together with 50 μg/mL of RNase  were subsequently added to respectively stain the nuclei and prevent staining of double-stranded RNA. After incubation for 5 min, the fluorescence intensity of an average of 2,000 nuclei per sample was analysed using a Partec CyFlow Space flow cytometer , equipped with a green solid state laser for PI excitation. The G1 peak of the standard was set to channel 720 and the amplification system settings were then kept at a constant voltage and gain throughout the experiment. For the data analyses, measurements were acquired using FloMax software v2.4d (Partec GmbH) in the form of six graphs: fluorescence pulse integral in linear scale (FL) (Graph 1); FL vs. time (Graph 2); side light scatter (SSC) vs. forward light scatter (FSC), both in logarithmic (log) scale (Graph 3); FL vs. fluorescence pulse height (Graph 4); FL vs. FSC (Graph 5) and FL vs. SSC (Graph 6). A region of interest comprising mostly the isolated nuclei was defined in the FL vs. SSC cytogram and subsequently used to gate all the other graphs. The presence of aggregates (doublets or triplets) was evaluated in the FL vs. fluorescence pulse height cytogram. The genome size  of each sample was determined according to the following formula: (mean PI fluorescence of the sample/mean PI fluorescence of the standard nuclei) * nuclear DNA content of the standard nuclei. The reliability of the genome size measurements was verified by evaluating the quality of the flow cytometry histograms based on the coefficient of variation (CV) of the G1 peaks and the background debris, and the CV of the genome size estimation of each isolate based on three independent measurements.The nuclear DNA content of For genotyping, the same individuals (gametophytic blades) used previously for flow cytometry were scored and DNA extractions were obtained from up to 4 sections of each blade referred above.30 as modified by31. Genotyping using 11 selected microsatellite markers33 generated 121 multilocus genotypes  automated capillary sequencer at CCMAR, Portugal, and sized with GeneScan-350ROX size standard.Genomic DNA was isolated using the LiCl extraction protocol described byx or 4x). Alleles were scored in GENEMAPPER v.4.1 (Applied Biosystems). Binning and allele rounding performed by GENEMAPPER v.4.1 was also checked with TANDEM34. The calculation of genetic diversity among the samples was estimated using GENODIVE, which allows analysis of polyploid data35. Summary statistics of genetic diversity within populations were calculated, including N: Number of alleles , Eff_num: Effective number of alleles , Hs: Heterozygosity within populations , also known as Gene Diversity, and Ht: Total heterozygosity, the expected frequency of heterozygotes over all populations, assuming Hardy-Weinberg equilibrium. Both Hs and Ht have a correction for sampling bias stemming from sampling a limited number of individuals per population. Each parameter was calculated with and without the maximum likelihood-correction for allele dosage in polyploids implemented in GENODIVE35.Calculation of genetic distances for all pairwise comparisons and estimation of genetic differentiation were performed on the whole data set and in 19 groups according to the ploidy levels found (see results below). Ploidy levels in genotypes were determined by flow cytometry and also by the maximum number of alleles found in a genotype for comparison. Number of alleles, allele range, and unbiased total heterozygosity were determined per group containing similar genotypes. The indices of heterozygosity are used as a theoretical index of genetic diversity; they do not represent observed heterozygosity. For mixoploids (blades containing more than one ploidy level) and samples with different types of cells mixed (e.g. zygotospores mixed with vegetative cells), the highest ploidy level of the cell lines found in the sample was considered. For multiploid gametes , the basic ploidy level found in the samples was considered (3ST/JostD) for all pairs of groups between the three species and among groups within each species  and they were also calculated with GENODIVE35.Genetic distance represents the amount of between-population difference in allele frequency summed over all loci. Genetic differentiation between individual loci was measured by pairwise genetic distances  was unknown and the population structure was inferred . The modal value of ΔK distribution for the posterior probability of the data for a given K was used as an indicator of the strength of the signal detected by Structure and considered as the real number of K clusters37. Each K was replicated 10 times with 10,000 Markov Chain Monte Carlo (MCMC) iterations after a burn-in period of 50,000, without any prior information on the ploidy and/or species group of each sampled individual. The average membership coefficients for the 10 simulation runs of a given K value were generated by CLUMPP v1.1.238 and a graphical presentation of the average membership coefficients for each isolate was generated in Microsoft Excel. An estimate of the true number of populations, K, was calculated using an ad hoc statistic-based approach implemented in STRUCTURE HARVESTER v0.6.139, as described previously. To avoid bias caused by missing values in the PCA and Structure analyses, missing data were replaced with random values randomly picked from the allele pool of each population as in40. Finally, in order to evaluate the degree of association between genotypic variation and ploidy levels, a neighbour-joining (NJ) network was generated from a matrix of pairwise41 genetic distances of individuals (genotypes), using POPULATIONS 1.242.In order to illustrate the variation in genetic diversity and genotypic differentiation (distribution of genotypes among the populations), a principal component analysis (PCA) based on allelic variation across all genotyped individuals was performed using GENODIVEAll the data (flow cytometry data set and genotypes) are available in supplementary information online (Appendices 1 and 2).P. linearis: 71, P. umbilicalis: 69, P. dioica: 77) using 122 blades . Considering this reference, the remainder genome size values obtained were assigned a ploidy level. Evidence from genotyping (next section) also indicated that gametophytes and gametes presented 2 or more alleles per locus, so being ruled out as haploid; thus, we assigned the gametes with the smaller genome size (0.2 pg) the first ploidy level as 2x. Eight different genome size values were obtained across the Porphyra samples, whose ratios could only correspond to seven ploidy levels shown in Table x, 3x, 4x, 6x and 8x were the most common among the samples. Two cell lines were associated with these ploidy levels: cell line 1: 2x–4x–8x and cell line 2: 3x–6x. In some samples of male gametes, two further genome sizes were obtained, 0.9 pg and 1.2 pg, which correspond with ploidy levels 9x and 12x. Genome sizes of 1.6 pg/1C corresponded to the G2-phase of ploidy levels 8x  Tetraploid blades 4x with 4x gametes; Group 2 in which gametophytes of each polyploid race (triploid or tetraploid) produced gametes of the same and/or different ploidy, (c) Triploid blades 3x with gametes 2x and/or 3x, (d) Tetraploid blades 4x with gametes of the same or different ploidy ; and Group 3 in which gametophytes belong to mixoploid races in which cell lines 1 and 2 were present in the same individual and they produce gametes of different ploidies also: (e) Mixoploid blades (2x/3x), (f) Mixoploid blades (3x/4x) and (g) Mixoploid blades (2x/4x). In general, for mixoploid blades or “Mx blades” (containing more than one ploidy level), we indicated each ploidy level separated by a “/” . When gametes with the same and lower ploidies were found in the same blade, we separated the different ploidy levels by a “,” . G1 and G2 on the top of each peak represent nuclei at G1-phase and G2-phase.In total, 7 different cytotype combinations were found in these ps Table . Group 1x, , and Mp gametes 4x . The 2x ploidy level was only found in gametes and in vegetative parts of mixoploids; no single blade was detected as exclusively diploid (2x).In addition, in Group 2, in the Tetraploid race,  and 8x (in 4x blades). In mixoploids, the zygotospores were only the double of the highest ploidy level . Furthermore, zygostospores (Zp) were found in either the marginal cells of the blade or in the vegetative part of the gametophytic blade for each polyploid race. When zygotospores and blades were sometimes found mixed with gametes (unfertilized); we indicated each of the ploidy levels of each cell separated by a “+” .The diplophasic phase 2Porphyra species, the incidence of each cytotype combination differed among them. In roughly, P. linearis was the species with the highest number of mixoploids (40%) and blades produced gametes of any ploidy level . Also, 17.5% of the blades were 4x with 4x gametes; 37.5% blades were 3x with 3x gametes, and a small proportion (5%) of triploids produced male gametes with lower ploidy. In P. umbilicalis, 40% of the individuals were 4x with 4x gametes, 17% were 3x with 3x gametes and 37% were either 4x or 3x that produced gametes with lower ploidy. Mixoploidy was also present, but in a much lower percentage (6%) than in P. linearis. For P. dioica: 42% were 4x with gametes 4x, 3% were triploid 3x with gametes 3x and 55% were fronds 4x with gametes of different ploidy levels.Although the overall average genome size did not differ among the three x, 4x, 6x, 8x), and “combined ploidy levels”   containing genotypes with different ploidy levels and different type of cells in the same sample.In total, 121 multi-locus genotypes were found (Appendix ST and Jost D (Table P linearis between triploids vs. tetraploids (GST/Jost D: 0.27/0.25) and triploids vs. mixoploids 3x/4x (GST/Jost D: 0.17/0.15). The rest of the group comparisons presented values close to zero. In addition, no differentiation was found between any groups in the other two species (data not shown).Genetic diversity, both measured as mean number of alleles per locus and the standardized effective number of alleles, was similar for the three species Table . When co D Table  was founP. linearis), indicating reproductive isolation mediated by ploidy. Within P. linearis, genotypes with different ploidy levels (triploids vs. tetraploids vs. mixoploids) were genetically differentiated. Also, the genotypes of the 2n phase, zygotospores 6x and 8x, were closely related to cytotypes 3x and 4x respectively. Within P. umbilicalis and P. dioica, no genetic structuring by ploidy level could be identified in the PCA . However, for P. dioica, there was not a clear structure among triploids and tetraploids. The STRUCTURE analyses separated well the three species but also showed genetic lineages within each species  found for P. linearis K = 2 (two clusters), P. umbilicalis K = 3 (three clusters), and P. dioica K = 2 (two clusters) , results were similar to the analyses of species separately, including distinct clusters for each species, and then distinct genetic lineages for each species. With further subdivision until K = 9 all groups different ploidy levels within each species were revealed  with all the genotypes together, the three species were strongly differentiated Fig. , as wereies Fig.  with besrs) Fig. . With alsee Fig. . ClusterPorphyra species studied are polyploid. Porphyra species showed high variability in genome sizes (eight), ploidy levels (seven), with two different cell lines found and at least three distinct lineages which comprised seven cytotypes. We show that the gametophytic phase (n) in Porphyra is not haploid, in disagreement with the assumptions derived from Drew21 landmark paper. Our results confirm the haplophasic/diplophasic cycle in Porphyra, with replicated and unreplicated nuclei, but not with the ploidy levels previously assumed, nor the simplicity of only two ploidy levels (haploid vs. diploid) assumed. The terminology haplophasic/diplophasic phase is already described for other plant species43.Our results show that the gametophytes of the three P. linearis, genetic differentiation between polyploid types (triploids vs. tetraploids vs. mixoploids) was strong; in P. dioica there was no significant genetic differentiation (most of the genotypes were tetraploid); and in P. umbilicalis, there was some degree of genetic differentiation (weak) which revealed discrete genetic clusters. Gene flow in polyploid lineages in these Porphyra species is likely mediated either with intermediate lineages (mixoploids) or by individuals that release gametes of lower ploidy levels that can fecundate different cytotypes. In P. dioica, the population is dominated by tetraploid individuals with gametes of multiple ploidy levels.There was important genetic differentiation and structure among the three species Figs –5. WithiPorphyra system is a complex system formed by autopolyploids, mixoploids and allopolyploids that interact among and between them. Triploids and tetraploids are normally originated by the fusion of gametes of different ploidies originated by irregular mitotic processes. Mixoploids are more likely originated by meiotic processes in the germination of the conchospore into four different tetrads  as reported before by previous authors for some Porphyra spp. (e.g.ref44), and consequently different cell lines within the same individual. We have not tested here the hypothesis of whether gametes from one species fecundate gametes from another species (with the same or different ploidy level), but we have observed some shared genotypes between P. dioica and P. umbilicalis , including algal species such as Gracilaria tikvahiae McLachlan48 and a Porphyra species: P. yezoensis50. In Porphyra yezoensis Ueda  M.S. Hwang & H.G. Choi) meiosis occurs as the conchospores germinate and the four resultant cells, which all have different genetic compositions, form the mature blade through successive divisions44. However, Niwa et al.52, have been reporting both chimeric blades and uniform blades, the last ones associated to asexual reproduction. In the species analysed in the current study, we have detected chimeric blades (mixoploids) and blades with one vegetative cell line (triploids and tetraploids), of which some individuals produced gametes of the same ploidy and others produced gametes of multiple ploidy levels. Three types of mixoploidy have been discovered: 2x/3x, 2x/4x, and 3x/4x. When analysing samples randomly collected in each blade, we always obtained the same result. Thus, it can be concluded that the formation of mixoploids for the samples examined was not sectorial, and the two different cell lines seem to be mixed within the blade. However, further research would be needed to study and confirm this aspect in detail.Genetic chimeras or organisms with more than one cell line have been reported for several plant species 53, or diploid sperm and haploid eggs (e.g. in plants)55. The mixoploids blades found here could reflect a long-term strategy in the Porphyra system of concealing and admixture of different cytotypes in the same species, preventing the exclusion of new cytotypes after emergence (similarly as the minority cytotype exclusion theory56). In the same way, blades that produce gametes of different ploidy levels act as bridges among cytotypes. The generation of gametes of lower ploidy  in tetraploid blades prevents the elimination of triploids and also ensures the admixture of different polyploid lineages.We hypothesize that in our polyploid system, mixoploids or blades with two cell lines are a bridge between ploidy types. In polyploid research, triploids are believed to act as bridges between ploidy types; in such a role, they mediate the formation of tetraploids from diploids or facilitate gene flow between diploids and tetraploidsPorphyra are yet to be described. Decrease in genome size has been reported to be adaptive. Rapid elimination of DNA sequences from autopolyploid genomes seems to occur in species such as Elymus elongatus, where both newly synthesized autopolyploids as well as natural accessions showed a loss of 10% DNA compared with their diploid progenitors57. However, this reduction is performed in a different way, since genome reduction is done in all the cells in the plant, not only the reproductive cells.We can’t answer how the production of gametes of different ploidy levels is performed. The processes involved in the DNA reduction in P. dioica  the only explanation to these sizes is the presence of spontaneous chromosome duplication/triplication phenomena. We discard the hypothesis of the presence of aggregates (doublets or triplets) since no changes in nuclei pulse width were detected among the nuclear populations, as expected if doublets or triplets existed (doublets show larger pulse width when compared with singlets). However, spontaneous chromosome duplication has already been reported in Porphyra species58 in59. However, as no blades of higher ploidy levels (>4x) were found, we presumed that those gametes are unviable. A simple explanation for this would be that since only male gametes presented those ploidy levels, fecundation with female gametes is not possible. Still, further research is needed to clarify this point.In the case of gametes of higher ploidies (“multiploid gametes”) found in Porphyra species in relation to the general assumption of a haploid/diploid life history , while life history 3 is typical of P. linearis, and present in P. umbilicalis.Based on our results, three new life histories are proposed for ory Fig. . In lifeory Fig. , two genory Fig. , both liy 3 Fig. , three lHaplospora globosa  possess the same number of chromosomes. However, the DNA level of sporophytic nuclei is twice that of gametophytic nuclei60. Another example is the gametophyte of Mediterranean Caulerpas, which can be diploid or triploid61. In Porphyra, incongruences in chromosome numbers have also been reported. For example, for some species of Porphyra, there is no change in chromosome number between the gametophyte vs. the sporophyte (e.g. refs65). However, if those species are exhibiting a parthenogenic life history, there is no explanation for the production and release of male gametes unless they were released to fecundate other polyploid races. We wonder now if the incongruences of the life histories reported in the literature for Porphyra spp. and other algal/plant taxa may be related to the recurrent presence of polyploid events and/or to the occurrence of mixoploidy undiscovered. And in that case, we believe that many algal groups have to be revisited and the life histories re-described according to the ploidy levels. Knowing that polyploids form at relatively high frequency  in flowering plants10 and also that formation of polyploids is possible in animals like fish, reptiles, amphibians and even mammals66, provides support to the hypothesis that many other polyploid algal species might remain undiscovered.The alternation of generations in algae and land plants typically describe the gametophytic generation as being haploid, and the sporophyte generation as diploid. However, there are many examples, which are not correlated with this haploid/diploid model. For example, the nuclei of sporophytes and gametophytes of the brown seaweed Porphyra species may be associated with the diversity of ploidy levels that we found. Since cryptic diversity in Porphyra has never been explored in this way, and also considering the evidence for genetic differentiation and isolation for the two polyploid races in Porphyra linearis, triploids and tetraploids, we believe they represent distinct evolutionary lineages with incipient speciation in P. linearis from the Iberian Peninsula, and therefore they may be considered distinct species. Future studies regarding the formal nomenclatural change in P. linearis are required.Our results suggest the hypothesis that the debate created on the cryptic diversity in Porphyra to study polyploid evolution. Since the gametophyte is the dominant phase and different cytotypes  coexist in the same population, the genus Porphyra can add novel insight into the evolutionary origins of polyploids. Interactions among these cytotypes can be studied in the same geographical region and hypotheses about intermediate cytotypes that may act as bridges can be formulated and assessed. The sister genus Bangia being the oldest eukaryotic multicellular taxon with sexual reproduction recorded67, also supports the interest of using the Bangiales to study other evolutionary questions unresolved such as, in an evolutionary scale, what was the first to occur: the gametophytes or sporophyte, and also may help to contrast hypotheses on the origin of the sporophyte and of the alternating generations in land plants68. Polyploidy is often associated to asexual reproduction, but in this case all the species studied are sexual, which is also interesting. Besides, many Porphyra/Pyropia species present asexual reproduction.The results of this study highlight the interest in using the genus 69 and many groups of vertebrates and invertebrates reviewed in refs73. Here we have reported a polyploid system with a high variability in ploidy levels, cytotypes and lineages with different life history strategies. Porphyra is a very interesting genus to continue the study of polyploid evolution, and towards the elaboration of evolutionary models for other red algae, and may be for other eukaryotes.Genome doubling (polyploidy) has frequently been associated with evolution and diversification in plants and has been a major factor in the evolution of other lineages of eukaryotes, including yeastPorphyra species. These represent two cell lines and comprise seven different cytotype combinations (with single and compound ploidy levels) among the same and different individuals. It is concluded that the gametophytic phase (n) in Porphyra is not haploid and more than one ploidy level is involved in this phase of the life history. In addition, three main distinct lineages for each Porphyra species are found: Triploids, Tetraploids and Mixoploids. In P. linearis, genetic differentiation was found among the three lineages: triploid and tetraploid, and mixoploid, representing different evolutionary units. Overall, the results of the present study reveal that Porphyra spp. constitute a complex polyploid system, composed by autopolyploids, mixoploids, multiploid gametes and probably spontaneous chromosome doubling, where three different types of life histories coexist but in different proportions depending on the species.In our study, many distinct ploidy levels (seven) and genome sizes (eight) were found in TABLE S1DATASET 1DATASET 2


To evaluate the effectiveness of different preventive programs in young adults at high caries risk using Cariogram software. Sixty-six young adults with high caries risk were evaluated. Dental caries risk for all subjects was determined according to WHO criteria. Subjects were divided into three different preventive groups . They were followed for 12 weeks . Plaque index, diet frequency, and salivary chairside tests  were performed at each visit. Based on these data, ten caries-related variables were collected and inserted into the Cariogram software to calculate the predicted chance of avoiding caries for each subject.p > 0.05). Significant changes were obtained about the Cariogram parameters . No significant differences were found between the three methods regarding mean Cariogram scores after 3 months ( The regular and effective short-term (three months) use of 1450 ppm fluoridated toothpaste, one visit application of fluoride, and chlorhexidine varnishes were effective for reducing caries risk in young adults, which can be clearly demonstrated using Cariogram software. Dental caries is still a major health problem worldwide with a multifactorial etiology, as it is due to the interaction of various factors including diet, the host's susceptibility, and the presence of microorganisms over a certain length of time . Along wNumerous caries risk prediction and evaluation models have been developed. They are all designed to evaluate the caries risk in a patient or a population as accurately as possible, but none has predominated over the others –5. AlthoThe aims of Cariogram are to (a) identify those persons who will most probably develop caries and (b) provide these individuals with the appropriate preventive and treatment measures to arrest the disease , 6. ThisDental caries can be prevented by applying suitable measures; hence it is very important to identify those individuals who are most likely to develop dental caries through caries risk assessment and to provide them with the required preventive measures to interrupt the disease process . There iHowever, to the best of our knowledge, no study has addressed the effects of different preventive programs on Cariogram parameters of high caries risk in young adults. Therefore, the aim of the present study was to assess consecutively the caries risk alterations of young adults at high caries risk following 12 weeks of three different caries preventive methods  using Cariogram.Ethical approval for this study was obtained from the Ethics Committee of the Dentistry School of the University of Selcuk (2012/10-22), and informed consent was obtained from all participants. A total of 246  young adults 18–25 years participated in this clinical study. Subjects were randomly selected in three different clinics during April 2012–September 2012.Carious defects, fillings, and missing teeth were diagnosed, and DMFT  scores were calculated according to WHO guidelines . Clinica∗Power software . A total sample of 66 subjects (22 per group) would give more than 80% power to detect significant differences with a 0.25 effect size between the three groups at a p = 0.05 significance level.The significant caries index (SiC) was calculated to select the one-third of the population with the highest caries scores. Therefore, 82 subjects were evaluated to be at high caries risk. The exclusion criteria for the patients were (1) systemic problem, (2) undergoing fixed orthodontic treatment, (3) smoking habits, (4) extreme plaque accumulation and periodontal problems, (5) no brushing habits, (6) taking antibiotics or any drugs in the last 3 months, and (7) regularly using fluoride mouthwash or other preventive methods. Sixteen subjects  were excluded from study. A power analysis was established by GThe study was carried out over a period of 12 weeks, with a total of four visits: first visit (baseline: T0), second visit (1 week: T1), third visit (4 weeks: T2), and final visit (12 weeks: T3). The visits were scheduled at the same time of day for each individual. Prior to each visit, the volunteers were asked to avoid tooth brushing and all other oral hygiene measures 24 h in advance and not to eat or drink anything but water for 1 h before the visit.All teeth that had carious lesions were restored with resin composite or amalgam. Each patient was given the same instructions with respect to oral hygiene. Using a small mirror with which the subject could also see the teeth inside the mouth, any plaque and gingival margins were shown to the subject and the importance of cleaning was well emphasized to them. All patients received the usual home-care oral hygiene instructions and a packet with nonprescription fluoride toothpaste (1450 ppm of fluoride), a manual toothbrush, and dental floss. The use of sugar-free chewing gum after meals was recommended for everyone. Each patient was given the same diet advice, such as reduction of daily intake . Potentially cariogenic foods and drinks  were described to the subjects. In addition, the results of the lactobacilli (LB) tests were shown and explained to the subjects at the first visit.n = 22 for each group): a control group (OH), fluoride varnish group (FV), and chlorhexidine varnish group (CV).Subjects were randomly allocated into three groups  three times a day during the 12 weeks after the first visit.FV. The participants received topical application of a 5% sodium fluoride  varnish . Firstly, the subjects' teeth were cleaned with a toothbrush. Secondly, excessive saliva in one or two quadrants of the mouth was removed by cotton rolls or by using an air syringe. It was not necessary to keep the tooth surface extremely dry because varnish could set in the presence of saliva. Thirdly, FV was applied to the tooth surfaces using a miniature cotton swab or brush, with the applicator dabbed repeatedly onto the tooth surface without contacting soft tissues. After a few minutes, a thin and clear layer was formed. Then the next quadrants were treated in the same manner. Patients were advised not to brush their teeth or chew food for at least 4 h after varnish application; during this time, soft food and liquid might be consumed. They were also told to use fluoridated dentifrices (1450 ppm F) for 12 weeks at home.CV. The individuals received a 1% chlorhexidine diacetate and 1% thymol varnish  application once a time after baseline measurement. Cervitec Plus application procedures were as follows: the tooth surfaces were cleaned thoroughly, dried with an air syringe, and isolated with cotton rolls. Three drops of Cervitec Plus were poured into a Dappen dish. A thin coat of varnish was applied by means of a Vivadent applicator on all surfaces of the teeth. The varnish was dispersed with air and allowed to dry, and the cotton rolls were removed after 30 seconds. Subjects were instructed not to rinse, eat/drink, or brush for one hour. They were also told to use fluoridated dentifrices (1450 ppm F) for 12 weeks at home.3, 1 = 103–104, 2 = 104–105, and 3 =>105 CFU/mL).Samples were collected in the morning between 9 and 12 a.m. under standardized conditions. Stimulated saliva was collected while chewing on a piece of paraffin wax for 5 minutes in a test tube graduated in milliliters, and the saliva secretion rate was expressed as milliliters per minute (mL/min). During collection, the patient was in an upright position. The buffer capacity of stimulated saliva was determined using the CRT Buffer® strip . After 5 min of reaction, a comparison was made with the colored chart provided by the manufacturer, and the buffer capacity was scored as low (pH below 4), medium (pH between 4.5 and 5.5), or high (pH over 6). The fresh saliva sample was then used to determine MS and LB counts. The CRT® Caries Risk Test (Ivoclar-Vivadent) was used to record the salivary counts of MS and LB. Both agar surfaces were wetted with saliva using a pipette without scratching the agar surface. The test vial was placed upright in the incubator (Ivoclar-Vivadent) and incubated at 37°C/99°F for 48 hours. After removal of the vial from the incubator, the densities of the MS and LB colonies were compared with the corresponding evaluation pictures in the enclosed model chart , were calculated. The Cariogram variables included in the statistical analysis were (1) diet (dietary content and frequency), (2) bacteria (MS level and plaque index), (3) susceptibility , (4) circumstances (caries experience and related diseases), (5) the predicted chance of avoiding caries, and (6) Cariogram risk levels. Diet, bacteria, susceptibility, and circumstances data were analyzed for statistically significant differences using Kruskal-Wallis and Wilcoxon tests. A one-way analysis of variance (ANOVA) and Tukey HSD tests were used to evaluate differences between the means for actual chance of avoiding caries for each group. A Friedman test was used for differences in Cariogram risk levels.The statistical analysis was processed with the SPSS 17.0 software system . A p > 0.05, 1The mean DMFT value was found to be 4.59 ± 3.43 for 246 subjects and 8.57 ± 2.17 for 66  study participants, respectively. No significant differences were found among the three groups regarding demographic properties . No significantly different MS levels were observed between the three groups on any visits (p > 0.05), except for T2 (p = 0.051). No significantly different LB levels were found between groups on any visits (p > 0.05) (p = 0.28). No significantly different buffer capacity, plaque index, diet frequency, or saliva secretion rate levels were found between groups on any visits (p > 0.05) .The only significant difference was found in the CV group for the MS levels during 12 weeks (t for T2 . A sligh > 0.05) . A numer Figures  and 4. AStatistical analysis revealed that the use of 1450 ppm F toothpaste, fluoride, and chlorhexidine varnish application resulted in a significant modification of the caries risk profile (Cariogram pie chart), thereby increasing the actual chance of avoiding caries in the future at each visit following T0. Tables 2p > 0.05). Regarding susceptibility level, a more prominent decrease was seen in the FV group than the OH and CV groups at T1, T2, and T3 (p < 0.05). These results were affected by fluoride program scores in Cariogram. The chance of avoiding caries increased significantly (p < 0.001) during the study for all subjects in the three groups separately: from 46.55 at T0 to 59.0 at T3 for the OH group, from 42.55 to 67.59 for the FV group, and from 42.09 to 60.27 for the CV group, respectively .No significantly different diet, bacteria, susceptibility, circumstances, chance of avoiding caries, or Cariogram risk levels were found at the four measurement times among the groups  and monitoring in three months recall periods could be considered as an average interval follow-up period for subjects in our study. Many studies have reported that a 12-week time-frame is sufficiently long to enable the semiquantifiable detection of preventive methods. Conversely, other studies suggest that a period of at least six months is preferable in order to detect any adverse or beneficial effects of caries preventive strategies. This study was limited to three months because of concerns that the high caries risk young adults, who were not familiar to regular dental examinations, would not comply with longer term follow-ups for personal and/or socioeconomic reasons.The only study conducted by combining the evaluation of the activity of preventive methods and the Cariogram program was performed by Mannaa et al., who evaluated the effectiveness of using 5000 ppm fluoride toothpaste using Cariogram for six weeks . In our 2 accumulation on the enamel surface. Fluoride varnishes developed for this purpose remain on the tooth surface for a longer period and slowly release fluoride to the oral environment  influence on caries risk is already established on the costuming settings of the program; therefore, the results can be biased.It is thought that the habit of brushing the teeth regularly and properly and the interdental plaque removal techniques must first be established, and regular implementation of the proper additional preventive applications in accordance with the individual's specific etiological caries risk factors is important for protection from caries and for reducing the individual to the middle or low caries risk group. The use of short interval control and application of caries risk tests, which motivated the patient about oral care, is one of the most important conclusions to be drawn from our study. The Cariogram program was effective and has some advantages such as making recommendation for preventive care and increasing patient motivation with its pie chart presentation so it can be used in the caries risk assessment instead of single variables.The regular and effective short-term (three months) use of 1450 ppm fluoridated toothpaste, one visit application of fluoride, and chlorhexidine varnishes were effective for reducing caries risk in young adults, which can be clearly demonstrated using Cariogram software.