Red foxes are a well-established species of urban ecosystems in the UK and worldwide. Understanding how foxes use urban landscapes is important for urban biodiversity and disease management. The Resource Dispersion Hypothesis (RDH) proposes that home range size is smaller in larger, better quality habitats and increases as such habitats become patchier. Here, we tested the RDH for foxes in the city of Brighton and Hove, focusing on predominantly residential areas, as foxes are reported to prefer residential gardens to other urban habitats. We compared fox range size according to extent of garden land cover and varying garden size and configuration. We tracked 20 foxes  with satellite collars recording at 15 min intervals for several weeks over four seasons to determine their range size and internal structure. We then measured garden size and configuration within fox ranges using Geographic Information Systems (GIS). We found that foxes had smaller ranges in areas containing few, large, well-connected gardens and larger ranges where numerous smaller gardens were crisscrossed by internal barriers  or fragmented by other habitats. Our findings confirm the RDH, showing that habitat dispersion can be used to predict fox movement in cities with relevance to urban planning.Vulpes vulpes) in the city of Brighton and Hove. We focused on residential garden areas, as foxes were associated with these in previous studies. We equipped 12 male and 8 female foxes with GPS collars recording at 15 min intervals during discrete seasons over four years. We regressed fox core area size against garden size, number of garden patches, and edge density within and between patches as extracted from GIS in a series of bivariate linear mixed models. We found that foxes used smaller core areas where gardens were large and well-connected and larger core areas where numerous, smaller gardens were fragmented by internal barriers  or bisected by other habitats such as managed grassland or built-up areas. Our findings confirm the RDH and help to inform future urban planning for wildlife. Red foxes are a well-established species of urban ecosystems in the UK and worldwide. Understanding the spatial ecology of foxes in urban landscapes is important for enhancement of urban biodiversity and effective disease management. The Resource Dispersion Hypothesis (RDH) holds that territory (home range) size is linked to distribution and richness of habitat patches such that aggregation of rich resources should be negatively associated with range size. Here, we tested the RDH on a sample of 20 red foxes ( Vulpes vulpes) [Angiostrongylus vasorum), causing angiostrongylosis in dogs, which is under-diagnosed and potentially fatal [Lyssa virus sp.) [Echinococcus multilocularis, which causes human alveolar echinococcosis [Increasing global expansion of urban areas typically has negative effects on biodiversity ,2, yet a vulpes) ,5. The p vulpes) , althoug vulpes) . Red fox vulpes) . Negativ vulpes) ,8, and r vulpes) ,10. Foxerus sp.) ,12 and tcoccosis . Attitudcoccosis , for exacoccosis ,15,16,17coccosis , territococcosis . In the coccosis ,20, Briscoccosis , Brightococcosis ).The Resource Dispersion Hypothesis (RDH)  providesUrban foxes are commonly associated with private residential gardens  patches can be quantified according to spatial scale such that, at the patch scale, a single garden separated from others by an edge  comprises a discrete patch. At the landscape scale, patches comprise an area of contiguous gardens bordered by one or more alternative urban habitat types. The number of patches at each scale and the extent (density) of the edge represent patch dispersion. In the current study, we explicitly tested three hypotheses in the context of urban ecology and the RDH. Hypothesis (H)1 (upon the acceptance of which H2 and 3 were contingent) was that availability of gardens should disproportionately affect the size of the home range relative to other urban habitats. Hypothesis 2 then held that the degree of fragmentation of patches at the garden scale  should be positively linked to fox home range size. In addition, patch size  should be negatively linked to fox home range size, because foxes need to use a larger area to access the resources required for reproduction where these resources are fragmented. H3 was a corollary of H2 and held that number of patches at both garden and landscape scales should be positively linked to range size, as the more numerous the patches, the greater the patch complementarity i.e., the likelihood that different key resources are available, in a heterogenous landscape. The study took place within the city of Brighton and Hove in East Sussex, United Kingdom . We sampled six residential zones encompassing a range of housing densities to representatively sample the existing variation in extent of man-made surfaces in residential areas . HousingLive capture took place between April 2012 and September 2015. We expended equal effort in capturing foxes in each of the six zones and aimed to achieve a balanced sample of females and males across the four seasons. Seasons were categorized as winter (December–February); spring (March–May); summer (June–August); and autumn (September–November). Foxes were captured in baited cage traps and fitted with tracking collars under anesthesia. Additional biometric data were collected during assessment prior to anesthesia or from anesthetized animals, including weight in kg, sex, body condition , and breeding status  if apparent . ApproxiAll collars were programmed to record location data via GPS satellites every 10–15 min, downloaded in the form of Universal Trans Mercator (UTM) Northing and Easting coordinates. Fox relocation data were “cleaned” by removing all “trap associated” data points (data from the time of release until 12 h afterwards) to exclude locations representing abnormal behavior following capture and anesthesia. To determine whether home range metrics were appropriate for our data , we implemented exploratory analysis in R v 3.4.0  using plug-in program rhr . We firsWe explored the use of various common home range estimators that describe the utilization distribution (UD)—a probability density distribution of animal relocations in two-dimensional space (see ). These Appreciating the importance of appropriate selection of smoothing parameters for accurate delineation of core areas in KDE , we perf2) and associated standard deviation (sd) and coefficient of variation (cv), number of patches (gardens), and edge density (ED in m/ha). Landscape scale variables were derived by eliminating the boundaries between adjacent garden patches using the dissolve tool in ArcGIS to make larger patches separated by other urban habitat types. At the garden patch scale, ED was calculated as the total length of internal boundary  per ha of total garden area as a measure of fragmentation between gardens. At the landscape patch scale, ED was calculated as the total length of boundaries with other urban habitat types per ha of total core area and was a measure of fragmentation of garden habitat by other urban habitat types. For both scales, ED excluded the edge generated by the border of core areas. We extracted the spatial variables relating to both gardens and other urban habitats from each of the three predominant land-use types in ArcGIS, calculated from Ordnance Survey’s Master Map (OSMM) topography and Open Greenspace datasets . The spa−1), and mean housing density squared (transformed to meet assumptions of linear regression) were controlled for via inclusion in additional bivariate models. Fox individual ID was included in each model as a random factor.To investigate the effect of spatial variables on fox core area size, we generated a series of bivariate linear mixed models (LMMs) in R v. 3.6.1  using the package lme4. The response variable (core area size) was log-transformed to stabilize variances and allow parameter estimation based on the assumption that the data were generated from a Gaussian distribution. The response was then regressed separately against a series of fixed factors to test each of the three hypotheses selected from an initial set after accounting for multi-collinearity (see Foxes were anaesthetized under UK Home Office license (PPL 7007429) under the guidance of a veterinary surgeon. Live capture, animal handling, and anesthesia were conducted according to a protocol approved by the Animal Welfare and Ethics Committee, University of Brighton. 2, and corresponding mean garden size was 0.012 ha (±0.01) or 126.3 m² (±102.1). The mean proportion of core areas covered by gardens was 0.43 (43% (±16)). Therefore, on average, a fox core area encompasses 451 gardens  = 1048 × 0.43). If we take the average housing density from our study of 44.6 (±22.9) resident households per hectare, one fox core area equates to 589 households (44.6 × 13.2). This is comparable to 451 gardens, as some of the households constituted flats without gardens. The proportion of core area comprising man-made surfaces was similar to that covered by gardens at 0.456 (45.6% (±13.3)) but more than four times larger than that covered by managed grassland (8% ± 6.6) or complex habitats with cover (1.6% ± 0.8). We GPS-tracked a total of 20 foxes (8 vixens and 12 dog foxes) in four seasons over a period of four years across three housing density categories . OverallF1,18 = 7.075, p < 0.05; F1,18 = 5.406, p < 0.05) and positively correlated with edge density , thus supporting H2, and number of patches at both garden  and landscape scales , thus supporting H3 . All other control variables  were non-significant  and landscape scale, although the latter correlation was weaker ; Core area size was negatively correlated with the ratio of garden area to combined other habitats  in the context of urban fox spatial ecology by investigating the relationship between fox core area size and residential garden patch size and dispersion. Our findings confirmed Hypothesis 1, as core areas were smaller where the ratio of gardens to all other habitats was highest. We inferred from this that gardens comprised the highest quality habitat relative to the other habitats available. We can also accept Hypotheses 2 and 3, as foxes ranged further where gardens were smaller, more fragmented (H2), and more numerous (H3). In addition, our findings indicate that fox ranging behavior is influenced not only by resource richness per se but also by uneven (patchy) resource distribution. This was due to the persistence of a strong positive association between core area size and number of patches at both the garden and the landscape scales, even after the variation attributed to fragmentation was accounted for. Fragmentation therefore negatively impacts habitat quality in some function of resource richness.However, foxes must also range over more patches, regardless of whether these patches are fragmented, because different patches provide different resources. To confirm this corollary of H3, it is necessary to directly quantify resources and demonstrate that resource heterogeneity is linked to patch configuration, which we strongly recommend for future work. Resources in urban gardens include supplementary food provided by householders, ponds, shrubs or mature tree cover for diurnal rest sites, and spaces under sheds and other outside structures for dens personal observations, ,16,44. FAlthough up to 78% of the variation in core area size was explained by number of patches at garden and landscape scales, differences between individual foxes contributed over a quarter (27.7%) of the variance. This indicates that fox-specific factors were influential in addition to environmental features. Intrinsic factors such as social organization and stage of lifecycle are important determinants of animal spatial ecology . FurtheIf we compare spatial statistics with other areas to determine representativeness of our sample, we see that the mean proportion of fox core areas comprising gardens in our study (42.8% ± 15.1) was similar to that reported by  in the UThe strong association found here between small fox ranges and a few, large, well-connected gardens is significant because fox social groups in urban areas are contiguous or overlapping where population density is high . Small, Our findings support the RDH in that fox space use was closely related to garden habitat patch dispersion, with implications for management of infectious disease, and urban biodiversity planning. Small fox ranges were associated with few, large, well-connected gardens. Further work is needed to identify the key resources in residential gardens and determine how foxes use them.


Polychlorinated biphenyls (PCBs) are human carcinogens, based on sufficient evidence for melanoma and limited evidence for non-Hodgkin lymphoma and breast cancer. Few data are available for liver cancer, although PCBs cause it in rats and determined liver damage in poisoned people. We investigated the association between PCB serum levels and hepatocellular carcinoma (HCC) with a case–control study in a PCB-polluted area in North Italy. We enrolled prospectively 102 HCC incident cases and 102 age and gender-matched hospital controls. Serum concentrations of 33 PCB congeners were determined by a gas chromatograph coupled to mass spectrometry. Of 102 HCC cases, 62 who had lost < 3 kg of body weight in past 3 years were included in the analysis . The odds ratio (OR) for HCC for 3rd compared to 1st tertile of PCB distribution was 1.76  for total PCB, adjusting for socio-demographic variables and risk factors for HCC by logistic regression. For most PCB congeners, ORs > 1.5 or 2 were found, although the 95% CIs included the null value for almost all of them. This preliminary study suggests that PCBs might play a role in HCC development. PCBs are included among the persistent organic pollutants (POPs) and accumulate in soil, plants and animals and along the food chain1. Experimental studies on animals and epidemiological studies on humans have demonstrated an association between PCBs and metabolic and endocrine diseases, damage to nervous systems and development and immunological and reproductive disorders2. Moreover, PCBs have been classified as “carcinogens to humans” (group I) by the International Agency for Research on Cancer, based on sufficient evidence of their carcinogenicity for melanoma and limited evidence for non-Hodgkin lymphoma and breast cancer2. For other sites and types of cancer, only sparse data were available.Polychlorinated biphenyls (PCBs) are a group of organochlorine compounds industrially produced until the 1980s, and banned afterwards in most countries due to their environmental persistence and toxicity1. Hepatotoxic effects were found also in humans with extremely high PCB intake in the intoxication incidents of Yusho and Yucheng1. An increased mortality from chronic liver disease and cirrhosis was observed in the Yucheng men, though not in women, in the early period after exposure3. Recently, an association between steatohepatitis and serum levels of various PCB congeners was found in a cross-sectional study on a residential US cohort with elevated PCB exposure4, and an association between PCB serum levels and unexplained ALT elevation was observed in a multicentre US cross-sectional survey5. PCB exposure causes liver cancer in rats, but occupational cohort studies showed inconsistent results as regards liver cancer mortality6, and no epidemiological study on the association between PCBs and HCC using biological measures of PCB exposure has been conducted so far, to our knowledge.The liver is a target organ of PCBs: animals exposed to PCBs show biochemical changes indicative of hepatocellular damage and liver dysfunction  and of fat liver deposition9. A study on a random sample of adults living in the town showed higher PCB serum concentrations than those usually found in people living in industrial area and not occupationally exposed, up to about 40,000 ng/g of lipid-adjusted serum total PCBs10.A chemical factory located in Brescia, the main town in the province, produced PCBs and other organochlorines from the 1930s to 1984 determining a heavy contamination of the soil, surface water sediments and locally produced food, including animal products 12. A case–control study carried out in 1995–2001 on 464 cases and 824 controls showed that hepatitis C virus (HCV), hepatitis B virus (HBV) and consumption of more than 60 g of ethanol per day for at least one decade were responsible for about 90% of the total cases occurring in the area14, in agreement with findings from other countries15. Also a role of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) in HCC development has been demonstrated recently17, but this condition seems still uncommon in our country, being responsible for about 5% of the total HCC cases in a recent large multicentre Italian study18. However, a role of other factors, including environmental toxics for HCC development, cannot be excluded. We carried a preliminary case–control study aimed to investigate the possible association between PCB exposure and HCC, taking account of the major risk factors for HCC in Western countries.Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for more than 80% of primary liver cancers worldwide. A high incidence of HCC has been registered in the Brescia area as compared to others in ItalyA total of 102 cases and 102 controls were enrolled. Among HCC cases, 40 said they had lost 3 kg and over of their body weight in the last 3 years. A comparison between HCC cases with and without recent weight loss showed that the former had significantly higher PCB serum levels than the latter: the medians of total serum PCBs were 1183 and 757 ng/g lipid, respectively (p = 0.02 by Mann–Whitney test). Among the 40 HCC cases with recent weight loss, no relationship was found between either quantity of, or time at, weight loss and PCB serum levels. Since HCC cases with recent weight loss may have higher PCB serum levels due to their release from lipid deposits into the blood, they were excluded from the analysis, which was therefore restricted to 62 HCC cases.Gender, age, residence and education and main risk factors for HCC in both cases and controls are shown in Table Age and gender distribution were almost identical between cases and controls, whereas some differences were observed according to residence, with 17.7% of cases and 35.3% of controls living in the town, and education, with 21.0% of cases and 36.3% of controls with more than 8 school years. Of the HCC cases, 41.9% had HCV infection, with or without other risk factors, 6.5% had HBV infection, with or without a history of heavy alcohol intake, 21% had a history of heavy alcohol intake alone, and 30.6% had no risk factors. As expected, few controls showed one or more of the main risk factors for liver disease: 4.9% had HCV or HBV infection and 4.9% had a history of heavy alcohol intake. A total of 44 cases (71%) had a clinical history of chronic liver disease with or without evidence of cirrhosis.The median, 90th percentile and range of lipid-adjusted serum concentration of all the PCB congeners and of the total PCBs in HCC cases and controls are shown in Table Of the 12 dioxin-like PCBs, only PCB 118 and PCB 156 were found, both at low levels. The PCB congeners found at the highest concentration in our study have endocrine or immunotoxic activities or can induce Phenobarbital . Slightly higher values of the median and 90th percentile were found in HCC cases than controls for all the congeners and total PCBs, although the differences were not statistically significant using the non-parametric Mann–Whitney test. A comparison between 44 HCC patients who had unifocal nodule and 18 cases who had multifocal or diffuse cancer was also performed, which showed no statistically significant differences between them for all congeners and total PCBs , although the difference was not statistically significant. No difference was found in PCB serum concentration according to residence in the main town vs rest of province, in both cases and controls, when adjusting for age.The distribution of HCC cases and controls according to tertiles of PCB concentration in the controls, and the corresponding odds ratios (ORs) adjusted for age, sex, residence, education and presence of one or more risk factors for HCC by multiple logistic regression are reported in Table No statistically significant difference of the ORs was found for 3rd compared to 1st tertile of PCB distribution for all PCB congeners (p > 0.1), and for each congener except PCB 194 (p = 0.02). ORs higher than 1.5 or higher than 2 for 3rd compared to 1st tertile were found for almost all PCB congeners, although they were not statistically significant. Accordingly, no statistically significant linear trend of OR increase with increasing PCB serum levels was found for total PCBs and for all PCB congeners except PCB 194.Similar results were found when restricting the analysis to subjects with, and subjects without, the main risk factors for liver disease Table : increasWe also performed subgroup analyses by gender, age and residence, which did not show substantially different results compared to the analysis of the whole dataset (data not shown in Table).19. In fact, we found that HCC cases who had lost more than 3 kg of body weight in the last 3 years, before HCC diagnosis, had higher PCB serum levels than those who had not lost weight, and we argued that higher PCB serum levels in the former were possibly due, at least partly, to mobilization of various chemicals, including PCBs, stored in body fat deposits. For this reason, we decided to exclude HCC cases with recent body loss of more than 3 kg.This preliminary study provides some evidence for an association between HCC and serum levels of PCBs, when also taking account of demographic and socio-economic factors and main risk factors for HCC, although the 95% confidence intervals of the estimates included the null value. These results are consistent for almost all the PCB congeners found in at least 30% of subjects. However, since HCC patients undergo liver cancer after a long history of cirrhosis, some concern may be raised whether the liver function impairment or the weight loss due to cancer development determine an increase of PCB serum levels in these patients20. HCC cases with and without the main risk factors for the disease showed similar PCB serum levels, and the ORs for HCC did not vary substantially stratifying subjects according to the presence or absence of at least one risk factor for HCC, suggesting that PCBs may be independent risk factors for HCC development.Most HCC cases had one or more major risk factors for HCC, namely HCV and HBV infection and alcohol intake > 60 g/day for at least 10 years, in agreement with a previous case–control study on HCC aetiology carried out in the same area2. The different PCB congeners, including dioxin-like and non-dioxin-like compounds, have different activities according to their chemical structure. We found that moderate and high chlorinated PCB congeners were detectable in most subjects and were at the highest serum concentration, particularly congeners 138, 153, 170, 180 and 194, in agreement with those most commonly detected in human studies2.The association between HCC and PCB exposure is biologically plausible as the liver is a target organ of these chemicals and an increased incidence of liver cancer is observed after their administration in rats2.However, PCBs usually occur in complex mixtures eliciting both genotoxic and non-genotoxic effects associated with carcinogenesis, tumour promotion and progression: in-vitro assays and experimental animal studies have shown that PCBs may produce oxidative stress and genotoxicity, interact with various receptors, including the aryl hydrocarbon receptor (AhR) and others controlling xenobiotic and steroid hormone metabolism or modulate plasma membrane-associated proteins affecting cell communication, adhesion and migration5, contrary to our findings of no relationship between PCB and ALT serum levels in the general population living in this area21. In the Anniston Survey, however, a cross-sectional study on a residential cohort with elevated PCB exposure, an association was found between serum levels of PCB congeners and those of the cytokeratin 18 biomarker, a more sensitive biomarker than ALT for environmental liver disease4.In humans, an association between serum levels of PCB congeners and unexplained alanine aminotransferase (ALT) elevation was found in the US NHANES22. However, almost 90% of the dioxin-like toxic equivalency (TEQ) in human blood of the contaminated people was contributed by PCDFs in both incidents23, arising doubts on the actual role of PCBs by themselves. On the contrary, although high levels of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) were also found in the general population in Brescia, the major contribution to TEQ serum levels was due to PCBs8. Some, but not all the occupational cohort studies found an increase in liver cancer mortality in PCB exposed workers with respect to national or regional figures24, but none of them had PCB serum measures.Few epidemiological data are available on the possible association between PCB exposure and HCC at present. The cohort studies of people who ingested rice oil contaminated with PCBs and polychlorinated dibenzofurans (PCDFs) in the Yusho and Yucheng incidents, in Japan in 1968 and in Taiwan in 1979, found an excess mortality from liver cancer and liver cirrhosis among intoxicated subjects in the first 10–15 years after the poisoning outbreak10.The main strengths of our study are that it was performed in a highly industrialized area with both high incidence of HCC and heavy exposure to PCB and possibly PCDD and PCDF as a consequence of half a century of PCB diffusion in the environment, with particularly high concentrations of these chemicals in soil, food and blood of residentsThis research has some limitations, too. Particularly, the limited number of HCC cases enrolled, due to the relatively high proportion of those excluded from the analysis for recent weight loss, which has reduced the study power of detecting an OR of at least 2 for 3rd compared to 1st tertile of PCB distribution. Therefore, our study can be considered as an explorative investigation on the association between HCC and PCB exposure.25. This finding was expected, since low serum values of total cholesterol and triglycerides are usually found in HCC cases, due to advanced cirrhosis and diet followed before or during hospitalization for the liver disease26. In HCC patients, the partitioning between adipose tissue and serum is close to 1:1 only when both are expressed on a lipid basis. Indeed, in a previous study of 101 HCC incident cases enrolled in the same area, we found high correlations between serum and liver (Spearman r = 0.79), serum and fat (r = 0.91), and liver and fat (r = 0.75) concentrations of single PCB congeners and total PCBs, using lipid-adjusted PCB measures27. The retrospective study design allows the comparison only of present PCB serum levels between HCC cases and controls, ignoring past exposures to these chemicals. However, PCB serum levels are usually considered a valid measure of PCB body storage, as shown by the high correlations between PCB serum and adipose tissue levels found in our and other studies31.The use of PCB serum level as a measure of the body burden of these chemicals in HCC patients may be of concern, due to their history of chronic liver disease. PCBs are highly soluble in lipids and are mainly deposited in adipose tissue and transported in the blood by lipids. HCC cases showed lower mean values of total cholesterol and triglycerides (133.5 and 85 mg/dl) than those found in the Italian population aged 65–74 years 14 and those reported by the Italian Cancer Registries32 and enrolled in a recent large multicentre Italian series18. We choose the controls among subjects hospitalized for traumatic causes, vascular surgery or minor surgery diseases. They showed similar PCB serum levels as the general population of the same age living in the town, as found in a recent survey21. Therefore, we are confident that no substantial selection bias exists in this study.As regards the selection of subjects, we enrolled both cases and controls by the same study base, i.e. residents in Brescia province, matched by age and gender, admitted to the same General Hospital. We collected blood samples of both groups at the same time and stored them for a short time before performing blinded laboratory analyses. The HCC cases were prospectively recruited among all the incident ones admitted to the General Surgery and Hepatology Divisions of the main hospital in the area, and they had similar characteristics as those enrolled in previous case–control studies on HCC etiology carried out in the same areaThis preliminary study provides some, though not conclusive, evidence that PCB exposure may play a role in HCC development, independently of other risk factors.We carried out a hospital-based case–control study. The study base was the population living in the province of Brescia and born in Italy. The study protocol established to enroll 100 HCC cases and 100 age- and gender-matched controls based on a-priori defined power of detecting an OR of at least 2 for 3rd compared to 1st tertile of PCB distribution at the threshold of p = 0.05 using two-tail statistical tests.The cases were recruited consecutively and prospectively in two Divisions  of the main General Hospital of the province. 96% of the eligible cases agreed to participate in the study. Overall, we enrolled 102 patients with a first diagnosis of HCC (incident cases) in 2015–2018, before they underwent any treatment for the disease. The diagnosis of HCC was based on computerised tomography and was confirmed by histological analyses in 63 cases.We selected 102 control subjects in the same age range and gender as the cases and without history of cancer or hepatic, endocrine or autoimmune diseases, who were admitted to the same hospital at the same time as the cases. When a subject suitable for the study according to the inclusion criteria was identified, he/she was invited to participate, and, in case of refusal, another individual was chosen and invited to participate. Overall, about 70% of the eligible subjects participated in the study. The enrolled controls were hospitalized for traumatic causes, vascular surgery or other minor surgery.34, the HCC cases were also questioned about the quantity of, and time at, weight loss in the past.Both cases and controls provided a fasting blood sample for laboratory determination and were interviewed at the hospital on demographic variables, weight and height, residential and occupational history, smoking habit and alcohol intake by the research personnel. Since a rapid weight loss may determine an increase of PCB serum levels20. Heavy alcohol intake was defined as consumption of 60 or more grams of ethanol per day for at least 10 years.Total alcohol intake was computed according to the average ethanol content of wine (12 percent by volume), beer (5 percent) and spirits (40 percent) and the frequency and quantity of alcoholic beverages consumed in the past. We considered the intake claimed by the subject during the decade in his/her lifetime with the highest consumption (“peak”), which had provided valuable results for evaluating the dose–effect relationship between alcohol intake and HCC occurrence in previous case–control studies carried out in this areaThe Ethics Committee of the area approved the project , and each participant provided a written informed consent. All research was performed in accordance with relevant guidelines/regulations.35: 28, 31, 52, 74, 77, 81, 99, 101, 105, 114, 118, 123, 126, 128, 138, 146, 153, 156, 157, 167, 169, 170, 172, 177, 180, 183, 187, 189, 194, 196, 201, 203, 206 and 209.The serum samples were stored at − 80° until and analyzed within 6 months since withdrawal. We investigated the following 33 PCB congeners, according to WHO classification36, using an Agilent Technologies 6890 N gas chromatograph coupled with an Agilent Technologies MSD 5973 . A PONA column  was used for chromatographic separation with carrier gas Helium. A 2 µL injection at 250 °C was performed by a 7683 Series Injector (Agilent Technologies) in splitless mode with a salinized injection liner .The PCB analysis was conducted following a previously defined analytical methodThe limit of quantification  varied among PCBs but was generally less than 0.1 ng/ml for each congener.PCB analysis was performed at the Laboratory of Occupational Hygiene and Toxicology, Brescia University, Italy. The Lab participated in inter-comparison programmes for toxicological analyses in biological materials , and fulfilled the requirements for congeners 28, 52, 101, 138, 153,180 in occupational and environmental medical fields T.The instrument was calibrated using the standard solutions PCB-Mix 20 10 µg/mL in Isooctane and the PCB No. 30 10 µg/mL in Isooctane . As external quality control, the certified control serum 10 A/B by G-EQUAS  was included in each analysis.37: total serum lipid (g/L) = 2.27 * serum cholesterol (g/L) + triglycerides (g/L) + 0.623.Total PCB serum concentration was calculated as the sum of the 33 PCB congeners. Since PCB concentration is influenced by the amount of serum lipids, the ratio of PCB concentration to the total lipid levels was computed (lipid-adjusted PCB concentration) and expressed as ng/g lipid. We calculated the total lipid concentration from cholesterol and triglyceride levels using the formula proposed by Phillips et al.10, as potential risk modifiers of the possible association between PCB exposure and HCC. The presence of HBV and HCV infections was evaluated by testing sera for HBsAg and anti-HCV, respectively, using commercial immunoassays (EIA).We also investigated the three main risk factors for HCC in this area, i.e. HBV and HCV infection and alcohol intakeThe distributions of total PCBs and each congener serum levels were examined using common statistical techniques for exploratory analysis. Due to the asymmetric non-normal distribution of PCB values, the median, range and 90th percentile are reported. The differences in PCB concentration between HCC cases and controls were evaluated using non-parametric methods, particularly the Mann–Whitney test for impaired data. PCBs serum levels also were categorized into 3 categories, according to the tertiles of their distribution in the control group. Then the odds ratios (ORs) for HCC were computed for total PCBs and each congener as a measure of association between exposure and disease, as it usually done in case–control studies, assuming that the OR is a reasonable estimate of the relative risk. The reference category for computing the ORs was the lowest level (1st tertile). The ORs were calculated adjusting for sex, age, residence, education and presence of one or more risk factors for HCC, as they are often associated with PCB serum levels in the general population and therefore they are possible confounders of the association between PCB serum levels and HCC, using multiple logistic regression. The confidence intervals were computed at the 95% level. Furthermore, a test for linear trend was performed using the Wald test on the coefficients based on PCBs as continuous variables.All the statistical tests were two-sided with a threshold of p = 0.05 for refusing the null hypothesis. The statistical analyses were performed using the STATA software for personal computer .Supplementary Information


Prunus persica (L.) Batsch] are among the most exported fresh fruit from Chile to the Northern Hemisphere. Fruit acceptance by final consumers is defined by quality parameters such as the size, weight, taste, aroma, color, and juiciness of the fruit. In peaches and nectarines, the balance between soluble sugars present in the mesocarp and the predominant organic acids determines the taste. Biomass production and metabolite accumulation by fruits occur during the different developmental stages and depend on photosynthesis and carbon export by source leaves. Carbon supply to fruit can be potentiated through the field practice of thinning , leading to a change in the source–sink balance favoring fruit development. Thinning leads to fruit with increased size, but it is not known how this practice could influence fruit quality in terms of individual metabolite composition. In this work, we analyzed soluble metabolite profiles of nectarine fruit cv “Magique” at different developmental stages and from trees subjected to different thinning treatments. Mesocarp metabolites were analyzed throughout fruit development until harvest during two consecutive harvest seasons. Major polar compounds such as soluble sugars, amino acids, organic acids, and some secondary metabolites were measured by quantitative 1H-NMR profiling in the first season and GC-MS profiling in the second season. In addition, harvest and ripening quality parameters such as fruit weight, firmness, and acidity were determined. Our results indicated that thinning  mainly affects fruit metabolic composition at early developmental stages. Metabolomic data revealed that sugar, organic acid, and phenylpropanoid pathway intermediates at early stages of development can be used to segregate fruits impacted by the change in source–sink balance. In conclusion, we suggest that the metabolite profile at early stages of development could be a metabolic predictor of final fruit quality in nectarines.Peaches and nectarines [ Prunus persica (L.) Batsch] are among the most important fruit crops with a world annual production of approximately 25 million tons  content , but it P. persica (L.) Batsch var. “Magique”] from the commercial orchard “Viveros El Tambo” located in El Tambo, O’Higgins Region of Chile (34°28′30.4″S 70°59′07.5″W) from August to December 2013 (first season), and from August to December 2014 (second season). Full bloom was on August 30, 2013, for the first season and on August 13, 2014, for the second season. For the first season, thinning was performed at 42 days after bloom (DAB), while for the second season, it was performed at 63 DAB. The thinning treatments comprised unthinned trees (UTH) that consisted in maintaining the whole fruit load resulting in a final ratio of six leaves per fruit and thinned trees (TH) that consisted in removing the fruit to the proportion 40 leaves per fruit, which is used in commercial orchard. Considering that branches of P. persica are autonomous concerning carbon assimilation (n = 12) every 7 days during the whole season in a randomized block design. Fruit growth was determined by measuring the equatorial diameter of 10 fruits per treatment weekly from 5 DAB until harvest at 118 DAB in the first season and from 27 DAB until harvest at 138 DAB in the second season. Once the fruits were harvested, they were stored at 20°C for 11 days until reaching the “ready to eat” stage.The experiments were performed using 4 year-old early harvest nectarine trees  (Sigma-Aldrich) was added and shaken for 30 min at 37°C. The content of each tube was transferred into a vial with a micro insert.The extraction of polar metabolites was carried out with the protocol described by m/z range were recorded at a scanning speed of 2.66 scan cycles per second. The MS ion source and quadrupole temperatures were 230 and 150°C, respectively.Metabolomic analysis was performed by gas chromatography–mass spectrometry (GC-MS). Data were obtained using the protocol described by Mass Hunter Data Analysis Software (Agilent Technologies) was used to deconvolute the chromatographic peaks. Identification was performed by comparing the peak retention and mass spectra to the NIST library in the quantitative method. Raw peak area data were corrected using the actual peak area of the internal standard, the sample fresh weight, and a quality control (QC) sample representative of all samples.–1 at 40°C. The elution protocol consisted on isocratic gradients of 100 mM NaOH for 25 min. Finally, a washing step with 20 mM NaOH for 10 min was performed. Sugar content  was determined by reference to a standard curve from 10 to 200 μM.The extraction of soluble sugars was performed following the protocol described by t-test for mean comparisons. For metabolite data, principal component analysis (PCA), partial least square regression discriminant analysis (PLS-DA), and clustering analysis with Euclidian distance were performed on the normalized data from NMR and GC-MS data using Metaboanalyst 4.0  than fruits from unthinned trees. This trend was maintained until the end of fruit development.Stage 2 (S2) of fruit development was characterized by pit hardening and little or no increase in fruit size, especially in early to mid-harvest nectarines such as “Magique” variety. End of S1 and start of S2 were determined based on the beginning of stone lignification (lignin becomes pink using phloroglucinol as stained in In stage 3 (S3), a second increase of fruit size occurred, which corresponded to the period of 90 DAB until harvest at 118 DAB for the first season, and of 100 DAB until harvest at 138 DAB for the second season. The larger differences in fruit diameter between the thinning treatments were observed in S3 in both seasons . In the The effect of thinning treatment on final fruit size was observed in the first season only  when fruP < 0.05). On the other hand, in the second season, no differences in fruit weight were observed between treatments , S2 (81 DAB), and harvest (118 DAB) in the first season. A total of 24 polar metabolites were quantified in all stages of development and thinning treatment (Metabolite profiles (mainly primary metabolites) were determined using reatment , S4. Thereatment . Similarreatment . Interesreatment , in agreIn order to determine how thinning treatment affected metabolite profile, a clustering analysis was performed based on the top 24 metabolites shown by ANOVA in the first season and visualized with a heatmap . HarvestA multivariate statistical analysis was performed with the data obtained from both UTH and TH samples, using detected metabolites as predictor variables and stage of development and thinning treatments as response variables. PLS-DA was able to explain 52.8 and 19.3% of total variance with the two components . In thisPrunus avium) and plum , it was shown that genotypic differences in final fruit size are primarily a function of cell number  and acid invertase  activities  when thePpeSUT1 expression mainly in thinned trees probably supporting apoplasmic sucrose unloading at harvest. We also observed higher sucrose content at ripening in TH fruits compared to UTH  should be performed at the beginning of fruit development to be effective in increasing final fruit size. We showed that when performed at the right moment, thinning affects the metabolite composition of fruit mainly in earlier stages of development and driving resources to sustain cell division that will impact final fruit size. Metabolite data revealed that sugar, organic acid, and phenylpropanoid pathways at early stages of development (S1 and S2 stages) can be used to segregate fruits impacted by the change in source–sink balance. In conclusion, we suggest that the profile of these metabolites in early developmental stages could be a metabolite predictor of final fruit quality in nectarines.The original contributions presented in the study are included in the article/AMA and MLV designed the research. MPC, LM, GB, and DA conducted field work, fruit phenotyping, and sampling. MPC, MM, CD, and AM performed proton NMR metabolite analyses. VL-C, CF, and RP performed GC-MS analyses and data analysis using Metaboanalyst 4.0. MPC, LM, GB, and MLV performed HPAEC-PAD analyses. MC and AMA wrote the original manuscript. MPC, VL-C, AM, RP, and AMA edited and reviewed the original manuscript. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TDRD genes can lead to sterility in males.Tudor domain-containing proteins (TDRDs) play a critical role in piRNA biogenesis and germ cell development. piRNAs, small regulatory RNAs, act by silencing of transposons during germline development and it has recently been shown in animal model studies that defects in , TDRD5, TDRD9 and TDRD12) in testicular biopsy samples obtained from men with obstructive azoospermia , as controls, and various types of non-obstructive azoospermia containing hypospermatogenesis , maturation arrest , and Sertoli cell-only syndrome  as cases. One-way ANOVA test followed by Dunnett’s multiple comparison post-test was used to determine inter-group differences in TDRD gene expression among cases and controls.Here we evaluate gene and protein expression levels of four key TDRDs (TDRD1TDRD genes in SCOS specimens. Also, the expression of TDRD1 and TDRD9 genes were lower in MA samples compared to OA samples. The expression of TDRD5 significantly reduced in SCOS, MA and HP specimens than the OA specimens. Indeed, TDRD12 exhibited a very low expression in HP specimens in comparison to OA specimens. All these results were confirmed by Western blot technique.The results showed very low expression of TDRDs could be very important in male infertility, which should be express in certain stages of spermatogenesis. Infertility remains a significant global burden . EstimatPIWI-interacting RNA (piRNA) proteins have recently been shown to play an essential role in male fertility and spermatogenesis in humans . The piRP-element–induced wimpy testis (PIWI) proteins specifically recognize piRNAs to mediate transposon silencing. The Tudor domain of Tudor domain-containing proteins (TDRDs) have been shown to directly bind to PIWI to help regulate this process via their dimethylated arginine (sDMA) or lysine residues , potentiGiven the current knowledge that piRNAs and PIWI genes are involved in spermatogenesis, and the potential role of TDRDs in this process, we sought to evaluate TDRD gene expression in testicular tissue of patients with azoospermia.One hundred and nineteen NOA and OA men admitted to the Abortion Research Centre, Yazd Reproductive Sciences Institute, were entered into this study. All subjects were undergoing bilateral testicular tissue micro-dissection (mTESE) operations to attain spermatozoa for intracytoplasmic sperm injection (ICSI). The study was approved by the local Ethics Committee and written informed consent was obtained from all subjects. Preoperative tests included karyotyping and Y chromosome microdeletion analysis, and the levels of serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. Patients were not receiving hormone therapy and all had primary infertility. No history of TESE and cryptorchidism were reported for any of the participants. Patients with cystic fibrosis, chromosomal abnormalities and Y chromosome microdeletion were omitted from the study. Subjects with normal spermatogenesis were included as the control group. The best control samples were men with proven fertility, and the problem was that they never referred to infertility centers for mTESE surgery, so we used samples with normal spermatogenesis as the control group.n = 29), lack of germ cells , declined number of spermatozoa , and incomplete maturation of germ cells . No significant differences in age, LH, FSH, and testosterone serum concentrations were found between the OA, HP, MA, and SCOS groups  and interpreted by a trained pathologist to classify the samples with normal spermatogenesis  based on the manufacturer’s protocol and then stored at − 80 °C. In-solution DNase digestion was accomplished to eliminate DNA contamination. The concentration and purification of RNA was specified using Nanodrop2000 spectrophotometer  and confirmed by agarose gel electrophoresis. Template cDNA was synthesized from 1 μg of whole extracted RNA with Revert Aid First Strand cDNA Synthesis Kit  using oligo dT and random hexamer primers simultaneously for each reaction in an Eppendorf Mastercycler Gradient .-∆∆CT for TDRD1, TDRD5, TDRD9 and TDRD12. The 2-∆∆CT parameter displays the expression fold of TDRD with respect to the housekeeping ACTB gene.Initial denaturation for RT-PCR started at 95 °C for 8 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s . The expression of TDRD1 and TDRD9 was significantly lower in MA samples than controls     staining showed clear differences between samples Figs.  and 2. Tt) Figs.  and 4. Tst) Fig. . The wesst) Fig. .Fig. 1aTDRD1, TDRD5, TDRD9 and TDRD12 genes in testicular tissue of NOA patients in comparison with OA patients.Spermatogenesis is a complex process, involving many cell type- and stage-specific transcription factors , 21. TraRecent studies in mice models have demonstrated that Tdrd5 gene plays an important role in silencing of LINE-1, a transposon member of the long interspersed nuclear elements (LINE), which is involved in assembly of polar granules in germ cells, chromatoid bodies, and spermatogenesis , 25. PreTDRD genes found in SCOS was a common observation in this study, suggesting that the transcription of these genes could be limited to germ cells. Due to the lack of germ cells in SCOS, it seems likely that the expression levels of TDRD genes in the SCOS samples would not be detected. However, we did detect very low levels of expression of these genes in some individuals, which could show the existence of small foci of spermatogenesis in these specimens or mixed patterns in NOA subtypes. Western blot analysis results confirmed the findings from qPCR, and demonstrated lack of TDRD expression in SCOS samples. Because the expression of the TDRDs genes was found to be very limited in the qPCR technique in SCOS specimens, but not in the Western blot technique, this difference could be attributed to the inherent nature of the Western blot technique, which does not reveal very low protein expression.The lack of expression of TDRD1 expression was significantly downregulated in the MA group versus controls. In Mili knockout mouse models, infertility of male mice has been shown to be due to stopping spermatogenesis at zygotene-pachytene stages. It has been revealed that hypermethylation of TDRD1 may lead to spermatogenic dysfunction [TDRD1 was related to a diminished risk of spermatogenic malfunction [TDRD1 gene had low expression in MA samples. The results of this experiment could confirm the role of TDRD1 in the early stages of meiosis process, likely to suppress transposons, which is essential for the progression of human spermatogenesis.We also found that function . Xiao-Bifunction  identififunction . WesternTDRD5 was significantly reduced in almost all cases  compared to controls. Mitinori Saitou et al. reported that loss of Tdrd5 leads to absence of spermatozoa, transposon depression and infertility [Tdrd5−/− mutations were arrested round spermatid and disorganized of chromatoid bodies [Another finding of this study was that the expression of ertility . Tdrd5−/d bodies . The resd bodies . TDRD5 cTDRD9, in collaboration with the HIWI2 (human homologue of PIWIL4), has a substantial effect on the synthesis of the piRNAs. In Tdrd9−/− mutants, the whole quantity of piRNAs were entirely reduced in testis [TDRD9 have a high risk of infertility due to transposon desilencing [HIWI2 gene rs508485 polymorphism and increased risk of spermatogenesis defects [TDRD9 and NOA [TDRD9 like TDRD1 showed a significantly decreased in the MA samples compared with OA samples. Western blot analysis results proved that the protein encoded by the TDRD9 gene had low expression in MA samples. It seems TDRD9 and TDRD1 have approximately the same role in human and other animals and they are very important for the meiosis process.The n testis . Faruk Hilencing . Two sep defects , 34. Ara and NOA . In presTDRD12 exhibited a very low expression in HP specimens in comparison to OA specimens. In addition, Western blot analysis results proved that the protein encoded by the TDRD12 gene was not expressed in HP specimens. TDRD12 is contributing to a process that leads to compaction of the sperm nucleus, histone-to-protamine exchange, which is essential for the differentiation of round spermatid into spermatozoa [The TDRD12 is indirectly related to the TDRD1 and MILI, because it has a different location than they . Jung-Jarmatozoa . TDRD12 rmatozoa .TDRD1, TDRD5, TDRD9 and TDRD12 genes in NOA and OA samples by qPCR and Western blot technique. Given the potential role for TDRDs in male infertility, our results suggest there is a need for further investigation to improve knowledge about the etiology of male infertility.To our knowledge, this study is the first to evaluate the expression of


Portal hypertension is a series of syndrome commonly seen with advanced cirrhosis, which seriously affects patient's quality of life and survival. This study was designed to access the efficacy and safety of selective esophagogastric devascularization in the modified Sugiura procedure for patients with cirrhotic hemorrhagic portal hypertension. n = 30) and the modified Sugiura group . The primary endpoint measurement is the postoperative rebleeding rate. Secondary endpoint measurements included free portal venous pressure, liver Child–Pugh score, liver volume, portal vein width and blood flow velocity, survival rate, quality of life, and dysphagia as well as other complications one year postoperatively. This trial is registered with ChiCTR, number ChiCTR2000033468.  Sixty patients with hepatitis B cirrhotic hemorrhagic portal hypertension and meeting the inclusion criteria were selected and randomly divided by using computer into the selective modified Sugiura group . In comparison with the MSP group, the Child–Pugh score of liver function in the sMSP group significantly increased  and the incidence of dysphagia was significantly reduced  one year after surgery. There was a statistically significant difference in the quality of life between the two groups. However, there were no statistically significant differences in free portal venous pressure , postoperative liver volume , postoperative portal vein width , portal vein flow velocity , 1-year survival rate , and other complications between the two groups.  There was no statistically significant difference in rebleeding rates within one year after surgery between patients in the sMSP and MSP groups ( Selective esophagogastric devascularization in the modified Sugiura procedure may not lower the incidence of rebleeding in the short term based on our findings. However, it may significantly improve quality of life of patients with cirrhotic hemorrhagic portal hypertension, improve liver function, and reduce postoperative dysphagia. Portal hypertension is a common clinical syndrome characterized by an increase in pressure gradient between the portal vein and the inferior vena cava. Chronic liver diseases including alcoholic or viral cirrhosis are the main causes of portal hypertension . ComplicCurrently, there are various treatment strategies for portal hypertension and related complications, including medical treatment, endoscopic treatment, interventional therapy, and surgery , 7. In gPortosystemic shunting and devascularization are the most commonly used surgical procedures for the treatment of portal hypertension. The former may further increase the risk of liver damage and hepatic encephalopathy due to decreased hepatic blood flow through the portal vein . The HasThis study was a single-center, randomized controlled trial to evaluate the efficacy and safety of selective esophagogastric devascularization in the modified Sugiura procedure. Selective esophagogastric devascularization is to preserve the esophageal branch of the left gastric vein, or the paraesophageal vein, and to selectively devascularize the lower esophagus perforating vein and gastric branches of the left gastric vein. The study was approved by the Ethics Committee of the Second Hospital of Lanzhou University, and informed consent was obtained from all participating patients. The entire trial followed the guidelines for good clinical practice in clinical trials.We recruited patients with cirrhotic hemorrhagic portal hypertension who were scheduled to undergo surgery at the Second Hospital of Lanzhou University between January 2014 and December 2017. Inclusion criteria were as follows: (1) patients with decompensated hepatic cirrhosis after hepatitis B, combined with splenomegaly, hypersplenism, and esophagogastric varices; (2) after the first or second esophageal and gastric variceal bleeding, and the bleeding was stopped by medical treatment for at least one week; (3) no history of endoscopic ligation or sclerotherapy prior to surgery, and no TIPS or other devascularization treatment; (4) no thrombosis in the portal vein and its branches by preoperative abdominal CT or ultrasound; (5) no liver cancer or other malignant tumors were found; (6) it was confirmed during the operation that esophageal veins did not directly enter the esophagus, and perforating veins clearly existed; and (7) adult patients who agreed to undergo the modified Sugiura procedure and sign informed consent.This study used a randomized study design, with the random sequence generated by using a computer. Patients who met inclusion criteria were randomly assigned to the selective modified Sugiura surgery group (sMSP group) or the modified Sugiura procedure group (MSP group) in a 1 : 1 ratio. Random assignment was performed after it was determined that the patient's esophageal vein did not directly enter the esophagus.2.3.1.Under general anesthesia in the supine position, an “L”-shaped incision was made at the ventral midline along the left rib margin. We (1) searched for and ligated the splenic vessels and performed splenectomy; (2) isolated the gastric proximal end close to the greater and lesser curvatures of the stomach and then disconnected and ligated the left gastric vein, posterior gastric vein, short gastric vein, left inferior phrenic vein, and accompanying arteries; (3) dissected the esophagus about 7 cm away from the cardia and disconnected, dissected, and ligated the esophageal vein, high esophageal branch, and esophageal perforating vein; (4) made a 2 cm vertical incision on the anterior gastric wall at the site of 3–5 cm away from the cardia and free of blood vessels; (5) used a #26 size tubular stapler for transesophageal transection anastomosis, approximately 3–4 cm away from the cardia. The vertical incision of the stomach was sutured. After it was determined that there was no active hemorrhage, a drainage tube was inserted and the abdominal cavity was sutured.2.3.2.Based on the modified Sugiura procedure, the left gastric vein and its branches were selectively treated . Namely,This mainly refers to patients with paraesophageal veins directly inserting into the esophagus. Those patients were excluded, and the paraesophageal veins inserting into the esophagus were directly disconnected and ligated during the operation. The remaining interventions were the same as described above. All patients were subcutaneously given low molecular weight heparin sodium 1 vial, Qd, on the second day after surgery. When the platelets normalized postoperatively, 100 mg of aspirin was administered orally daily.Follow-up was scheduled at 1, 3, 6 months, and 1 year after surgery. Follow-up measurements included routine blood examination, liver function tests, color Doppler ultrasound of the portal vein and hepatic artery, venous color Doppler ultrasound, abdominal CT, quality of life assessment, and complications.The primary endpoint measurement is postoperative rebleeding rate. Secondary endpoint measures included free portal venous pressure (FPP), liver Child–Pugh score, liver volume, width of portal vein and blood flow velocity, quality of life, one-year survival rate, and various complications .For rebleeding, the main postoperative manifestations were hematemesis or hematochezia, diagnosed by gastroscopy. Free portal venous pressure (FPP) was assessed as follows: after laparotomy, FPP was measured three times before splenectomy, after splenectomy, and after transesophageal anastomosis. An indwelling needle with a diameter of 0.7 mm was used to puncture and fix the right gastroepiploic vein, which was about 10–15 cm away from the cardia. The indwelling needle was connected to a detector  with sensors . Quality of life one year postoperatively was measured by the SF-36 scale. Liver volume was calculated based on abdominal enhanced computational tomography (CT) and measured by the IQQA(R)-Liver Image Analysis System. Portal vein ultrasound was used to measure portal vein width and thrombus.t-test was used for continuous variables, and the chi-squared test was used for the two categorical variables. The difference between groups was considered statistically significant at a p value of less than 0.05.Statistical analysis was performed using SPSS version 18.0 software, and all analyses were intention-to-treat (ITT) analysis. The n = 30) or modified Sugiura procedure group (MSP) (n = 30)  ((n = 30) . There w(n = 30) . The aveχ = 0.11, p=0.73).None of the included patients developed esophagogastric hemorrhage during postoperative hospitalization. Six weeks after surgery, two patients in the sMSP group developed hematemesis and melena; gastroscopy diagnosed esophageal and gastric variceal rebleeding; four patients in the MSP group developed rebleeding. In the sMSP group, five patients had esophageal and gastric fundus hemorrhage within 1 year after surgery, of which one patient died of rebleeding 3 months after surgery. The remaining patients were treated with medical or endoscopic therapy with much improvement. The rebleeding rate was also 16.7% (5/30). A total of six patients in the MSP group developed esophagogastric hemorrhage within 1 year after surgery. The rebleeding rate was 20.0% (6/30). No death was reported. There was no statistically significant difference in rebleeding rates between the two groups 1 year after surgery ; The FPP value decreased slightly after transesophageal esophagectomy in comparison to after splenectomy; this difference was not statistically significant .In the sMSP group, the FPP value was significantly lower after splenectomy (20.01 ± 4.50 mmHg) and esophageal transection (19.40 ± 4.40 mm Hg) than before splenectomy (27.00 ± 6.41 mmHg). There was a statistically significant difference between the two groups .In the MSP group, the changes in the patient's FPP value were same as the sMSP group . There wA, 7 cases; grade B, 19 cases; and grade C, 3 cases. The Child–Pugh scores of the patients in the MSP group were as follows: grate A, 3 cases; grade B, 16 cases; and grade C, 11 cases. The differences between the two groups were statistically significant .There was no statistically significant difference in the Child–Pugh score of liver function between the two groups prior to surgery . One yeap=0.24; MD = −320.12, 95% CI: −438.43 to 102.78, p=0.16) (There was no significant difference in liver volume between patients in the two groups before surgery . The stu p=0.16) .p=0.50; MD = 0.17, p=0.21). No statistically significant difference in portal vein flow velocity was observed between the two groups before surgery .There was no significant difference in the width of portal vein between patients in the two groups before surgery . There w surgery . There wp < 0.001), general health (p < 0.001), social function (p < 0.001), and mental health (p < 0.001) in comparison with those in the MSP group. The differences between the two groups were statistically significant . In addition, in those patients who did not meet the inclusion criteria, one patient died of a chest infection following postoperative esophageal anastomotic fistula. The patient was treated with endoscopic sclerotherapy for esophageal and gastric variceal bleeding 1 month before surgery.One patient in the sMSP group died of esophageal and gastric variceal rebleeding 3 months after surgery. The 1 year survival rate was 96.66%. There were no deaths reported in the MSP group. No statistically significant difference between the two groups was observed . Ascites was a frequently encountered complication after surgery. The incidence of ascites in the sMSP group and MSP group was 83.33% and 90.00%, respectively. Ascites usually occurred 1 week after surgery and dissipated within 2 weeks of treatment. The high-risk incident time was 1 week to 3 months after surgery. Patients generally had no abnormal discomfort. Although patients in this study were given subcutaneous low molecular weight heparin sodium and oral aspirin prophylactically after surgery, the effect was not plausible. The incidence of postoperative abdominal hemorrhage in the sMSP and MSP groups was 16.6% (5/30) and 20.0% (6/30), respectively. There was no statistically significant difference between the two groups . Hepatic encephalopathy and esophageal anastomotic leakage were also critical complications. Although their respective incidence was not high, the treatment was challenging and the prognosis was poor.In addition to hemorrhage after surgery, postoperative complications also included ascites, portal vein thrombosis, splenic vein thrombosis, dysphagia, abdominal bleeding, abdominal infection, esophageal anastomotic leakage, and hepatic encephalopathy . In partPortal hypertension caused by cirrhosis is a common clinical syndrome that seriously affects the quality of life of patients. Liver transplantation is regarded as the only “curative” treatment of portal hypertension ; howeverA selective esophagogastric devascularization procedure was proposed by Yang et al. . SpecifiThe modified Sugiura procedure is a relatively complicated operation; it mainly applies to these patients with hemorrhagic portal hypertension. Indications of the surgery mainly include the decompensated stage of cirrhosis after hepatitis, accompanied by recurrent gastrointestinal bleeding, and the bleeding should be stopped before surgery. At the same time, liver function is an important index to evaluate the feasibility of surgery; Child–Pugh C may be a contraindication for performing the surgery .Our results show that selective esophagogastric devascularization in the modified Sugiura procedure can significantly improve the quality of life of patients 1 year after surgery, improve the Child–Pugh score of liver function, and reduce the incidence of postoperative dysphagia. However, different from our expectations, our findings did not show that sMSP was effective in reducing the risk of postoperative rebleeding, even though it did not increase the associated risk. In addition, we found that splenectomy had a remarkable effect on reducing FPP, while selective esophagogastric devascularization did not demonstrate a significant effect on FPP in a short term. It is unknown whether selective esophagogastric devascularization can affect FPP or how it affects FPP in a certain period after surgery. Based on the angle of portal vein pressure change, the benefit of modified Sugiura procedure or selected modified Sugiura procedure to patients was mainly obtained by splenectomy. It may have little to do with esophagogastric devascularization or esophageal transverse anastomosis. Nevertheless, it is certain that selective esophagogastric devascularization does not increase postoperative complications of the modified Sugiura procedure in the short term.Johnson et al.  and ZhanFor portal hypertension treatment, most treatment strategies including TIPS can be called “palliative,” except for liver transplantation. Therefore, the quality of life of patients appears to be a key indicator to assess the therapeutic effects. We showed that selective esophagogastric devascularization in the modified Sugiura procedure significantly improved the quality of life of patients 1 year after surgery. Such improvement in quality of life may be highly related to the improvement of postoperative liver function and the low incidence of dysphagia. However, it is still unclear whether the improvement in patients' quality of life was associated with the esophageal transverse anastomosis. What effects the esophageal transverse anastomosis may have on the postoperative quality of life of patients and how selective esophagogastric devascularization in the modified Sugiura procedure could affect quality of life of patients by abandoning the esophagus transverse anastomosis are questions that should be explored in the future.In conclusion, for patients with cirrhotic hemorrhagic portal hypertension, sMSP did not reduce the incidence rate of rebleeding in the short term based our finding. However, it may lead to significantly augmented quality of life in patients, improved liver function, and reduced postoperative dysphagia in comparison with MSP.


Fusarium oxysporum f. sp. cubense (Foc) race 1, is a major disease of bananas in East Africa. Triploid East African Highland (Matooke) bananas are resistant to Foc race 1, but the response of diploid (Mchare and Muraru) bananas to the fungus is largely unknown. A breeding project was initiated in 2014 to increase crop yield and improve disease and pest resistance of diploid and triploid East African Highland bananas. In this study, eight Mchare cultivars were evaluated for resistance to Foc race 1 in the field in Arusha, Tanzania. In addition, the same eight Mchare cultivars, as well as eight Muraru cultivars, 27 Mchare hybrids, 60 Matooke hybrids and 19 NARITA hybrids were also screened in pot trials. The diploid Mchare and Muraru cultivars were susceptible to Foc race 1, whereas the responses of Mchare, NARITAs and Matooke hybrids ranged from susceptible to resistant. The Mchare and Matooke hybrids resistant to Foc race 1 can potentially replace susceptible cultivars in production areas severely affected by the fungus. Some newly bred Matooke hybrids became susceptible following conventional breeding, suggesting that new hybrids need to be screened for resistance to all Foc variants.Fusarium wilt, caused by the soil-borne fungus  Banana cultivation in East and Central Africa (ECA) is dominated by a group of cooking bananas commonly referred to as East African Highland bananas (EAHB). These bananas were introduced into the region from Asia about 2500–3000 years ago, where after secondary evolution took place in the highlands of Uganda and Tanzania ,2. TodayEAHBs comprise diploid and triploid bananas ,7,8,9,10Fusarium oxysporum f. sp. cubense (Foc), is present in most banana-growing regions of ECA [Fusarium wilt of banana, a disease caused by the soil-borne fungus s of ECA . Foc infs of ECA ,17. Thiss of ECA . Foc pros of ECA ,17,18. Ts of ECA ,21,22,23s of ECA .Foc is diverse and comprises three races based on their pathogenicity to a group of differential cultivars, with Foc races 1, 2 and 4 causing disease to Gros Michel, Bluggoe and Cavendish bananas, respectively ,26. All Bananas can be evaluated for Fusarium wilt resistance in pot assays under screen house and greenhouse conditions, or in the field. Results obtained from pot and field screenings are not always similar. Field screenings provide an evaluation of plant response under prevailing environmental and geographic conditions, but depend on the distribution of inoculum in the soil and the Foc strain present . Pot assIn 2014, a project to breed EAHB for higher yields and for disease and pest resistance started in Uganda and Tanzania. Mchare hybrids were derived from crosses between Mchare cultivars and diploid bananas resistant to Foc race 1 . NineteeAll Mchare cultivars and Sukari Ndizi, the susceptible control, developed internal rhizome symptoms of Fusarium wilt in the screen house 10 weeks after inoculation with Foc race 1 with disease incidence between 88.9–100% . The disMakyughu II, Mchare Laini, Akondro Mainty, Mchare Mlelembo, Huti Green Bell and Ijihu Inkundu developed internal rhizome discolouration in the plant crop at Arusha, with a RDI that did not differ significantly from that of the susceptible control, Sukari Ndizi . Huti WhThe RDI rating obtained for the screen house and field evaluation of Mchare cultivars was significantly positively correlated for the first ratoon (r = 0.76) . This inThe Muraru cultivars were all susceptible to Foc race 1 with disease incidence between 88.9–100% and disease severity ranging from 3.7 for Muraru M3 to 5.8 for Njuru, compared to the 6.0 for the susceptible Sukari Ndizi . DiseaseNineteen NARITA hybrids were evaluated in the screen house. Of these, only NARITA 16 and NARITA 12 developed Fusarium wilt symptoms (disease incidence between 55.6–88.9%), with disease severity significantly higher than those in Mbwazirume, the resistant control, but also significantly less than those in Sukari Ndizi, the susceptible control . NARITA Sixty Matooke hybrids were evaluated for resistance against Foc race 1. Thirty-eight of these were considered resistant, as their disease severity did not differ from that of Mbwazirume, the resistant control. Among the 38 resistant hybrids, 25 hybrids did not develop any Fusarium wilt symptoms (disease incidence 0%), whereas 13 hybrids had a few plants displaying internal spots in the inner rhizomes (disease incidence between 11.1–83.3%), with an average RDI below 2 on a scale of 6 . SixteenUntil recently, a limited amount of information was available on the response of diploid EAHB bananas to Foc race 1, despite the seasonal appearance of Fusarium wilt on Mchare and Muraru bananas in Tanzania and Kenya, respectively ,34. ThisThe results obtained from pot and field evaluations were well correlated, indicating that the pot assay can be used for the mass screening of banana varieties. Results obtained from pot trials when using the millet seed technique reflected those from field evaluation. The millet seed technique has also been widely used for the mass screening of banana varieties for Fusarium wilt resistance ,43,44. F1 offspring. An advanced screening of F2 population could confirm the nature of the inheritance of resistance. Among the Mchare hybrids tested, hybrids obtained from the same parents  did not produce the same results. T.2274-6 and T.2273-2, for instance, were resistant, while T.2269-2 and T.2691-15 were susceptible to Foc race 1. Calcutta 4 is resistant to Foc race 1  and HutiMatooke bananas are resistant to Foc race 1 ,32. NARIThe disease development in the Mchare cultivars Kahuti and Huti White at the field site in Arusha was slow compared to other Mchare cultivars. Makyughu II, Mchare Mlelembo, Akondro Mainty, Huti Green Bell and Ijihu Inkundu developed Fusarium wilt in the plant crop, but Kahuti and Huti White only showed internal rhizome symptoms during the first ratoon. It is unlikely that these varieties escaped the disease in the first production cycle, as diseased plants had been spread in the field during trial preparation. The delayed disease development might, however, be attributed to low inoculum levels or the uneven distribution of inoculum in the soil. If the cultivars were not evaluated for at least two production cycles, they would have been ranked as resistant. It is therefore important to consider increasing the inoculum in the field and/or to evaluate two production cycles when evaluating banana for disease resistance in the field. The evaluation of internal rhizome symptoms of Fusarium wilt near harvest proved to be of greater value than the rating of external leaf symptoms. Kahuti and Huti White varieties displayed yellowing of leaves that were rated as Fusarium wilt symptoms in the plant crop, but transverse cutting of rhizomes near harvest time showed no disease. This suggests that leaf yellowing may have been caused by other stresses. The rating of internal rhizome symptoms to assess Fusarium wilt is therefore needed when trial material presents little or no external symptoms. The leaf symptoms of Huti White and Kahuti that were visible in plant crop were probably caused by other stresses such as drought, deficiencies or nematodes, as the resistant control Nakitengwa also displayed a level of leaf yellowing.In future, Mchare and Matooke hybrids with Foc race 1 resistance should be evaluated for agronomic performances and resistance to other important banana pests and diseases. Resistant diploids can also be used in banana breeding programs as sources of Foc race 1 resistance. Foc race 1-resistant EAHB hybrids should also be screened for resistance against Foc TR4. Foc TR4 has first been detected in northern Mozambique in 2013 , and poses a serious threat to banana production in neighbouring Tanzania. It is known that banana cultivars respond differently to Foc race 1 and TR4, and that Foc TR4 has a larger host range ,43,49,50Eight Mchare cultivars and eight Muraru cultivars were evaluated for resistance to Foc race 1 in Arusha , togetheFoc isolates CAV 3733 and CAV 3856, collected from infected bananas in Kawanda and Arusha, respectively, were used in Uganda and Tanzania as inoculum. The isolates were identified with Foc Lineage VI-specific DNA markers and by vegetative compatibility group (VCG) analysis. The Foc isolates were identified as Foc VCG complexes 0124/5/8/22 (CAV 3733) and 0124/22 (CAV 3856), respectively, which both belong to Foc race 1. The isolates are stored at the culture collection of Stellenbosch University’s Department of Plant Pathology. Eleusine corocana) seeds were washed and soaked in tap water for 6 h, the water was drained and the millet seeds autoclaved in 415 × 600 mm autoclavable bags  on two consecutive days for 20 min at 120 °C. Twenty to 30 mycelial plugs (10 mm × 10 mm) from a Foc race 1 isolate, grown on PDA for 4–5 days at 25 °C, were then used to inoculate 3 kg of sterile millet seeds in the autoclaved bags. The bags were incubated in the dark at 25 °C for 10–14 days and shaken every third day to ensure that the fungus properly colonised the millet seeds. A millet seed technique was used to inoculate banana plantlets in screen house trials . Finger For screen house inoculations, 2-month-old banana plants were inoculated with Foc-colonised milled seed mixed into the potting soil. The eight Mchare cultivars, as well as the Matooke and NARITA hybrids, were planted in plastic pots containing 2 kg potting soil mixed with 20 g of the inoculum. The Muraru cultivars and Mchare hybrids, however, were replanted into 1 kg potting soil inoculated with 25 g of Foc-colonized millet seed. Before the greenhouse trials were conducted, a pilot study showed that the two inoculum concentrations gave the same result. After inoculation, the experiments were set up in a completely randomized block design (CRBD) with three plants per block, replicated three times. Thus a total of nine plants were used for each accession tested. In Arusha, Tanzania, Grand Naine  and Nakitengwa (EAHB) were used as resistant controls for the evaluation of Mchare cultivars, and Sukari Ndizi as the susceptible control . For theFor the screen house experiments plants were considered ready for rating when 50% of the susceptible control plants displayed leaf symptoms. This corresponded to a period ranging from 10–12 weeks after inoculation. During evaluation the rhizomes were cut open and the discolouration of inner rhizomes scored on a rating scale ranging from 1–6 .Field evaluation of Mchare banana cultivars for Foc race 1 resistance was conducted in Arusha, Tanzania. The land used was naturally infested by the fungus, and additional inoculum was added before the trial began by cutting up banana plants with Fusarium wilt symptoms, and spreading the diseased plant tissue across the trial site. Eight Mchare cultivars were then planted in a CRBD in April 2017, with 18 plants per cultivar in each block, replicated three times. . NakitenDisease in the field was monitored for two production cycles, known as the plant crop and the first ratoon. Disease progression in the field were scored using a 1–5 rating scale based on leaf discoloration . The plaAn analysis of variance (ANOVA) was performed to compare Fusarium wilt resistance of the banana genotypes evaluated in the screen house and field trials, based on their RDI. XLSTAT software  was used to compute ANOVA, and multiple comparisons were performed between variables according to Fisher’s test at the 0.05 level of least significance difference (LSD). Pearson correlation test was performed between screen house and field screenings of Mchare cultivars in Arusha. The genotypes were ranked as ‘resistant’ when their RDI was less than ‘2’, as ‘partially resistant’ when it was between 2 and 3, and as ‘susceptible’ when the RDI was more than ‘3. To compare the progression of leaf yellowing in the field trial in Arusha, Tanzania, the AUDPC was measured  and an Aix is the severity of the disease observed at time it, ix+1 the severity of the disease at the time of the subsequent evaluation i + 1, it the time (months) at the time of observation i; it+1 the time (months) at the time of the subsequent evaluation i + 1, and n the total number of evaluations. The AUDPC was calculated as follows: AUDPC =