The EU Water Framework Directive (WFD) requirement that all surface water bodies achieve good ecological status is still a goal for many regulatory authorities in England and Wales. This paper describes field and laboratory studies designed to identify metal contaminant loadings and their distributions within water bodies located in the Lower Lee catchment . Water and sediment samples have been collected from increasingly urbanised sites on the River Lee and its main tributaries over a two-year period with samples analysed for total concentrations of cadmium, copper, lead, mercury, nickel, tin, and zinc. Complimentary batch tests indicate a positive relationship between aqueous metal concentrations and the batch test-derived sediment metal release data, particularly during wet weather events. Field data indicate a dynamic relationship between water and sediment concentrations with both being capable of exceeding relevant environmental quality standards/sediment quality guidelines at all sites. Mean sediment metal concentrations across all sites were found to be highest for Cu (141.1 ± 111.0 µg g The European Union (EU) Water Framework Directive  establisThe Lower Lee catchment has a long history of water quality problems as a consequence of increasing urbanisation associated with factors, such as discharges from both point sources (wastewater treatment plants (WWTPs)), industry, commercial enterprises), diffuse sources , navigation, and water abstraction ,5. WasteIn polluted waters, many contaminants are predominantly adsorbed to suspended particles in the water column and to sediments settled on the river bed. This identifies sediments not only as pollutant sinks but also as potential sources of contamination as a result of changes in environmental conditions and/or anthropogenic disturbances. Sediments represent a more stable medium for tracing metal sources compared to water . WhereasSediment quality criteria are not specifically identified in the existing legislation associated with the Water Framework Directive , but theRivers represent key resources in terms of providing water for drinking supply, industry, and crop cultivation. Additionally, in urban catchments they are required to receive wastewater and surface runoff discharges whilst maintaining recreational and conservation facilities. This paper investigates the ability of the lower River Lee, an important urban river in North East London, to fulfil these functions through a study of the spatial and temporal trends in the metal levels in both sediments and the overlying waters. Seven metals  have been monitored to obtain a better understanding of how sediment-water interactions can influence their distribution and hence their impact on the urban river environment.2 in which the geology consists of London clay with deposits of alluvium and river gravels, overlying chalk. Consequently, the river levels can peak rapidly during high rainfall events leading to the potential for flooding. The waterways within the Lower Lee catchment serve as sources of water supply for London, recipients for treated sewage discharges, navigation channels, as well as providing recreational and environmental resources. The catchment drains an area of 367.4 kmWater and sediment samples were collected from five progressively urbanising sites  guidelines . The tes−1, 29.4 ng L−1, 223.7 ng L−1, 75.1 ng L−1, 7.3 ng L−1, 22.9 ng L−1, and 47.7 ng L−1 for Cd, Cu, Hg, Ni, Pb, Sn, and Zn.Water and sediment samples were subjected to microwave digestion  according to an adaptation of US EPA methods 3015A  and 3051g for 10 min, the supernatant was transferred to a volumetric flask, and together with washings, made-up to a volume of 100 mL with deionised water. The metal concentrations of all sediment samples were determined using ICP-OES . Instrument detection limits were 0.3 ng g−1, 0.8 ng g−1, 0.8 ng g−1, 0.4 ng g−1, 1.0 ng g−1, 1.3 ng g−1, and 0.2 ng g−1 for Cd, Cu, Hg, Ni, Pb, Sn, and Zn. Using a certified reference material (SQC001 (Lot 011233), Sigma-Aldrich, Poole, UK), recovery efficiencies from sediment samples of between 81.1% for Ni and 96.5% for Zn were determined. Multi-element calibration standards of 0.1, 0.5, 0.75, and 1 mg L−1 were prepared for sediment analyses with the six standards for water analyses covering the concentration range of 2–20 µg L−1. All analyses were carried out in triplicate and daily performance checks were conducted on each instrument prior to commencement of analyses. The instrument calibrations were checked every 10 samples throughout the analytical procedure by running one of the calibration solutions as an unknown. Recalibration was performed if drifts in the calibration measurements exceeded 10%. The precision of the methodology (% RSD) for water extractions was typically <15% for all metals, but improved to < 5% for sediment extractions, except for Sn where it was <10%.Sediment samples (accurately weighed to approximately 0.5 g) were extracted in pre-cleaned PTFE-TFM digestion vessels using concentrated (70%) nitric acid (9 mL) and concentrated hydrochloric acid (3 mL). An analytical reagent blank was prepared using the acids only. After microwave digestion and subjecting to a centrifugal force of 3060× The concentrations of selected metals in surface waters collected at the five sites over a two year period are shown in Two of the eight sampling collections occurred following wet weather conditions . The highest mean concentrations of Zn, Ni and Sn (with the exception of site A for Sn) were monitored on these occasions (May 2015 (4 mm) and August 2015 (15.2 mm)). In contrast the highest mean concentrations of Cd (except at Site A) and Cu were consistently reported during a dry weather sampling event  indicating that the catchment is in receipt of pollutants during both dry (point source) and wet weather (diffuse) events and that some pollutants may be preferentially associated with specific sources. These results highlight the complexities associated with identifying pollutant sources in an urban catchment ,31,32.−1 (for DO), from 8.1 to 8.7 (for pH) and from 12.9 to 15 °C (for temperature), with no parameter showing a consistent trend by site location or sampling date (data not presented). Comparisons with UK TAG water quality guidelines [EU environmental quality standards (EQS) are available for Cd, Hg Ni, and Pb, and UK WFD Technical Advisory Group (TAG) standards  exist foidelines  for riveAn overview of the concentrations of selected metals in surficial sediment at each of the five sites is reported in In the absence of national or international environmental quality standards for sediments, r = 0.59; p ≤ 0.05) indicating that metals stored in sediments at this site are potentially more susceptible to release. Greater potential for the release of metals from sediments at this site could also indicate that sediments are a source of the elevated aqueous metal concentrations also reported at site B  exists. Further examination of the sediments regarding their mineral and total organic content composition, sediment pH and cation exchange capacity may reveal the processes driving this relatively higher release for other metals at this site, but these were not within the scope of the current study.All metals (with the exception of Hg) show the greatest release at site B ); see Analysis of the mean water and sediment metal concentrations did not reveal a statistically significant relationship between the two environmental compartments. This demonstrates the complex relationship that exists between metal behaviours within the two matrices. However, whilst mean aqueous metal concentrations did not appear to vary significantly in relation to mean sediment metal concentrations see , the corr = 0.928; p ≤ 0.05) and Pb , and for Hg and Sn during the May wet weather event . Significant positive correlations were also determined during some of the dry weather events , November 2014 (Hg), December 2014 (Cd), and March 2016 (Hg). Whilst the overall pattern is not clear or consistent for all metals, the data does support the suggestion that the aqueous metal water concentrations are influenced by the releasable sediment metal concentrations (as determined from batch tests) during both wet and dry weather conditions in the field. The enhanced flow volumes and flow rates associated with wet weather conditions will encourage the resuspension of sediment and, consequently, the release of previously particulate-associated metals [Likewise, positive significant correlations were also evident during the August wet weather event for Cu (d metals , as has Analysis of water and sediment metal concentrations showed that tributaries originating from heavily urbanised environments contribute to the heavy metal load of the River Lee. Metals transported into the river are likely to be stored in the sediment and, as shown by the batch tests, can be released into the overlying water, resulting in exceedances of water quality standards. The proportions of metals released varied with different weather conditions and site specific parameters. Zinc and Hg were found to be released most frequently, based on the correlation between sediment release potential and water concentrations, with concentrations of Zn consistently exceeding the UK TAG standards during correlated events and are, therefore, of particular concern. The results of this study highlight the importance of monitoring sediment as well as water quality and further analysis of sediment properties, water parameters, and chemical speciation of metals can strengthen the capacity of predicting pollutant release from contaminated sediments into the overlying water body. The findings are of relevance to all urban rivers in receipt of point and diffuse pollution, and of particular pertinence to those requiring remediation to achieve the environmental objectives of the EU Water Framework Directive .


Mutations of ACTN4 cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS). Presentation usually occurs in the teenage years or later with symptoms of mild proteinuria and slowly progressive renal dysfunction leading to end-stage renal disease (ESRD). We report a 5-year-old female patient who was diagnosed with nephrotic syndrome and did not respond to 6 weeks of oral glucocorticoid therapy. Renal biopsy showed a collapsing variant of FSGS and genetic studies revealed a heterozygous disease-causing mutation in the ACTN4 gene . No mutations were found in the NPHS2, TRPC6, and INF2 genes, nor did her parents have any mutations for FSGS. She developed ESRD 6 months after presentation. Although a disease-causing ACTN4 mutation was identified, the contribution of additional polymorphisms in other genes is not known. Such additional polymorphisms may represent yet unidentified epigenetic factors that contributed to the aggressive nature of this child’s disease progression. A literature review has revealed only two similar case reports.  Mutations in the gene encoding alpha-actinin-4 (ACTN4) are the leading causes of autosomal dominant form of focal segmental glomerulosclerosis (FSGS) accounting for ~ 2 – 4% of cases of familial FSGS . MultiplPatients with mutations in ACTN4 typically present in the teenage years or later and have slow progression of renal dysfunction. Some may progress to end-stage renal disease (ESRD), usually in the fifth decade of life , 4. This report illustrates a case of a collapsing form of FSGS secondary to sporadic ACTN4 mutation that rapidly progressed to ESRD. A literature review revealed one case report of two siblings with p.Ser262Phe mutation in the ACTN4 gene and p.Thr5Met substitution in NPHS1 gene (of unknown significance). Both patients presented with NS in early childhood, one of whom rapidly progressed to ESRD. Renal biopsies showed collapsing glomerulopathy in one patient and FSGS not otherwise specified in the other. Their father, who was clinically silent, possessed a germline mosaicism of the mutation [3/µL with 87% segmented neutrophils and 5% bands, normal serum electrolytes, a serum creatinine concentration of 0.68 mg/dL, and a serum albumin concentration of < 1 g/dL. The urinalysis was positive for 4+ proteinuria and moderate blood, but no red cell casts were seen. A CT scan revealed bowel wall thickening and free fluid in the abdomen. At the time of admission, a blood hemoglobin concentration 9 g/dL was recorded and the blood cultures were positive for Streptococcus pneumoniae. The patient was diagnosed with bacteremia secondary to peritonitis from new onset nephrotic syndrome and was started on glucocorticoid therapy along with a course of intravenous cefotaxime followed by oral levofloxacin. A 5-year-old Caucasian female presented with abdominal pain and emesis. She had experienced facial puffiness for 2 weeks prior to onset of other symptoms. On examination, she was found to be febrile with tachycardia, anasarca, and described diffuse abdominal tenderness. Laboratory studies showed an elevated white cell count of 38.5 × 10The patient showed no response after six weeks of daily glucocorticoid therapy. A renal biopsy revealed variable degrees of wrinkling and collapse of the basement membranes within thirteen of the 32 glomeruli as well as occlusion of the capillary loops. Two of these glomeruli were globally sclerosed. Proliferation of visceral epithelial cells was apparent . One cor2. Blood pressure was controlled with a regimen of amlodipine, furosemide, losartan, and metolazone. Six months after presentation, the patient progressed to ESRD and started on peritoneal dialysis for hyperkalemia, acidosis, and fluid overload. After being managed on peritoneal dialysis for 5 months, she underwent a living unrelated donor transplant and has not experienced a recurrence of disease. DNA sequence analysis was performed by PCR amplification of highly purified genomic DNA, followed by automated bi-directional DNA sequencing of the coding regions of the NPHS2, ACTN4, TRPC6, and INF2 genes. Of the INF2 gene, which has 22 exons, exon 1 is non-coding and thus was not included in the analysis. Additionally, only the 3’ end of exon 8 was analyzed. In exons where one of the strands was not informative for confirmation, a uni-directional sequencing approach with alternative dye chemistry was used for confirmation. At least 20 bases of intronic DNA surrounding each exon were also sequenced. Genetic studies showed a heterozygous disease-causing mutation in the ACTN4 gene   with no Podocytes are terminally differentiated and highly specialized epithelial cells that cover the outer layer of the glomerular basement membrane (GBM). The podocyte and mutations/polymorphisms in podocyte-specific genes are central to the pathogenesis of FSGS . PodocytFSGS is defined by involvement of some portion of the tuft  of some glomeruli  in sclerosis accounting for ~ 20% of all the causes of nephrotic syndrome in children . FSGS isIn 2000, Kaplan et al.  discoverWe did not perform a functional study of the p.Ser262Phe substitution in ACTN4 as this same amino acid substitution has been described in prior studies. Both parents did not have mutation of ACTN4 gene and were asymptomatic, suggesting that this variant was truly disease causing. The unique features of this case are the presentation in early childhood, the presence of a collapsing form of FSGS, and the rapid progression to ESRD. ACTN4 mutations usually manifest in adult life, though interestingly, not all persons carrying disease-associated ACTN4 mutations develop clinical disease. It has been suggested that a second hit, representing either a second mutation in another gene or a physiological stressor may induce a disease process in susceptible persons , 12. TheThe authors have no conflicts of interest to disclose


Countless studies have identified differences between the gut microbiota of humans affected with myriad conditions and healthy individuals, and animal models are commonly used to determine whether those differences are causative or correlative. Recently, concerns have arisen regarding the reproducibility of animal models between institutions and across time. To determine the influence of three common husbandry-associated factors that vary between institutions, groups of weanling mice were placed in either static or ventilated microisolator caging, with either aspen or paperchip bedding, and with one of three commonly used rodent chows, in a fully crossed study design. After thirteen weeks, samples were collected from multiple regions of the gastrointestinal tract and characterized using culture-independent sequencing methods. Results demonstrated that seemingly benign husbandry factors can interact to induce profound changes in the composition of the microbiota present in certain regions of the gut, most notably the cecum, and that those changes are muted during colonic transit. These findings indicate that differences in factors such as caging and bedding can interact to modulate the gut microbiota that in turn may affect reproducibility of some animal models, and that cecal samples might be optimal when screening environmental effects on the gut microbiota. Conversely, the composition of the GM can be affected by the health status of the host, often leading to the question of whether differences observed between healthy and affected individuals in the GM composition are causative or merely correlative3. Thus, a comparative medicine approach employing animal (often rodent) models for prospective studies with longitudinal sample collection is vital in this rapidly growing area of research. Despite the common goal of optimal health status and elimination of pathogens, the husbandry of laboratory rodents is not however standardized. In addition to the obvious differences between barrier-housed and conventionally housed rodents, there are many subtle differences within either type of housing including, but not limited to, bedding type4; commercial source, formulation, and post-manufacture treatment of food5; air flow (static versus ventilated)7, commercial source, and overall size of caging; and housing density8. The influence of these often overlooked variables on the composition of the GM is largely unknown and there is the potential that husbandry-induced changes in the GM could contribute to a lack of reproducibility between labs or institutions10.The collection of microorganisms present in the gastrointestinal tract (GIT) of multicellular animals, often generically referred to as the gut microbiota (GM), represents a complex and diverse ecological system with an undeniable influence on host health and disease susceptibility11, energy utilization13, and the density and composition of the luminal microbiota14. It is thus reasonable to believe that disease or treatment-induced effects on the GM may go undetected in studies based purely on fecal samples. Considering the regional differences in the recognition of, and response to, the gut microbiota via mucosa-associated lymphoid tissue (MALT)16, any such undetected differences may very well be of physiological relevance. Thus, we hypothesized that differences in husbandry such as bedding and caging type or dietary formula would result in changes in the intestinal microbiota, and that those differences would be more evident in certain regions of the GIT.Additionally, the majority of research involving the GM, whether performed in humans or animal models, focuses on the fecal microbiota as the necessary samples can be acquired noninvasively, thus allowing for longitudinal studies with repeated measures. There are however distinct differences between regions of the GIT with regard to overall tissue function, cell type distributionn = 12/group) one week after arrival at our facility and again 12 weeks later. Moreover, to determine whether any changes detected in the composition of the fecal microbiota following 12 weeks under the different conditions were representative of the microbiota present in other regions of the GIT, samples were also collected at necropsy of the luminal contents of the jejunum, ileum, and cecum. Microbial communities were characterized via 16S rRNA amplicon sequencing using the Illumina MiSeq platform and the influence of caging, bedding, and diet on various aspects of the GM were tested using permutational multivariate analysis of variance (PERMANOVA)18 and three-way ANOVA via a general linear model, as appropriate.To determine the influence of three common husbandry variables  on the composition of the GM of laboratory mice, a fully-crossed longitudinal study design was devised, incorporating two different bedding types (compressed paper and aspen chips), two different caging types , and three different dietary formulations purchased from the same source . These are all commonly used research rodent diets, the primary differences being irradiation of LabDiet 5008, and macromolecular content. Specifically, 5008, 5053, and 5058 contain crude protein of at least 23%, 20%, and 20%, and crude fat of at least 6.5%, 4.5%, and 9%, respectively. Fecal samples were collected from 144 mice  of the baseline fecal samples. Perhaps reflecting the relatively young age of these mice at the time of arrival, the fecal profiles, resolved to the level of OTU, were somewhat inconsistent both between and within treatment groups at one week after arrival , bedding (p = 0.760), or diet (p = 0.050) on richness were detected  and allowed to acclimate for one week prior to collection of a baseline fecal sample. Twelve weeks later, representing a common study duration in contemporary research, mice were euthanized and luminal contents of the jejunum, ileum, and cecum, and an end-point fecal sample were collected. Following extraction of DNA, PCR was used to generate libraries of amplicons representing the V4 region of the 16S rRNA gene. These libraries were sequenced using the Illumina MiSeq platform, resulting in amplification of 138 of 144 baseline fecal samples (95.8%) to a threshold of 5000 sequences. Based on independent rarefaction of samples from each sample site and time point , coverage of greater than 5000 sequences resulted in coverage-independent estimates of richness . Pairwise comparisons within endpoint fecal samples based on the Bray-Curtis distances however, when corrected for multiple testing, detected only two significant differences between groups (out of 66 comparisons) in fecal community composition , with F values ranging from 36.71  to 207.8 . Comparison of the richness of samples from each region revealed a similar division between the upper and lower GIT with a mean (±SEM) of 58.9 (±1.4), 66.0 (±1.0), 45.7 (±0.4), and 48.1 (±0.3) OTUs detected in the jejunal, ileal, cecal, and endpoint fecal samples respectively  and lower  GIT along principal coordinate 1 (PC1) which described 28.78% and 20.45% of the variation in the Bray-Curtis Fig.  and Jaccely Fig. . KruskalLactobacillus sp. , unidentified microbes in the order Clostridiales, family Lachnospiraceae, and Turicibacter sp. While there were several similarities between ileal and jejunal contents, the brief transit time between regions nonetheless resulted in distinct microbial populations. OTUs that were generally reduced in relative abundance in ileal contents, relative to the jejunum, include Lactobacillus sp. and microbes in the order Streptophyta  in the candidate genus Arthromitus  were intermittently detected at a high relative abundance of several ileal samples. These microbes are probably more properly classified as Candidatus Savagella, the genera of SFB found in mammalian hosts21. PCoA of jejunal samples demonstrated variable clustering depending on the distance matrix used , and Oscillospira sp. while these taxa were detected at much lower relative abundance in the aforementioned mice , we also found no differences in fecal microbiota of wild type mice housed in different settings. However, we found unexpected stark differences in the cecal microbiota of mice subjected to different housing conditions. Moreover, the observed differences in the cecal microbiota were dependent on an interaction of two variables, cage ventilation and bedding, and no differences were detected in the cecal microbiota of mice exposed to only one of the two factors in question. One possible explanation for this bedding/housing interaction effect is the presence of an unknown aromatic or volatile compound in aspen bedding that is removed from ventilated microisolator cages but remains in static cages at a level capable of influencing the cecal microbiota. Any such compound could be exogenous in nature or may be of host-origin, such as urinary ammonia absorbed differentially by the two types of bedding. In addition to removal of volatile compounds, the increased noise or airflow of IVCs have also been shown to be a potential stress factor for mice23 which may influence GM composition. Collectively, our findings in conjunction with those of previous studies22 highlight the complexity by which factors  may influence intestinal microbiota.The data presented above carry several implications for animal modeling, particularly in the context of research investigating the gut microbiota. At the most basic level, our findings provide proof-of-concept that seemingly benign factors associated with rodent husbandry can influence the composition and heterogeneity of the resident microbiota in certain gut regions. Thoene-Reineke 24, and corncob, hardwood, and paper bedding containing high, moderate, and low coliform counts respectively4. Those findings may partially explain differences between mice housed on hardwood and cotton bedding in total IgA production, although the specificity of the enhanced mucosal immune responses by mice housed on hardwood bedding is not clear25. In combination with other data suggesting the potential of microbial contamination of corncob bedding, these findings suggest that corncob and hardwood beddings may be more likely sources of environmental microbial contamination than paper or cotton products. Similarly, numerous studies have investigated the impact of housing density29, cage change frequency30, caging system and ventilation32, and other husbandry-related variables from the standpoint of animal well-being but very few have explored the influence of these variables on the GM composition.Comparisons of coliform counts and lipopolysaccharide (LPS) levels in various types of rodent bedding, performed using standard microbiological techniques and fluorometric assays respectively, suggest that there are biologically relevant differences, with hardwood and corncob beddings containing significantly greater levels of LPS relative to paperThe present data also suggest that cecal contents may be a better indicator of environmental influences on GM, and the use of fecal samples may lead to “false negatives” when screening for effects on the GM. The reason for this is unknown but the microbial composition within the lumen of the GI tract apparently normalizes during colonic transit. While the use of feces as the representative sample provides several obvious benefits such as non-invasive acquisition , collection and analysis of cecal contents should also be considered in terminal studies, particularly those investigating the effects of environmental influences on the GM. Alternatively, longitudinal studies incorporating analysis of cecal communities could be performed using separate cohorts of mice taken down at intervals.34, particularly studies employing animal models, these and other data35 demonstrate the need to consider the gut microbiota, and by extension, the husbandry of research animals. Researchers at different institutions performing the same experiments, with the same strain of mouse purchased from the same vendor, could generate different data and reach ostensibly different conclusions. Both could be completely valid datasets, the difference merely reflecting a microbial influence on experimental outcomes. In addition to the differences in the composition of the cecal microbiota between mice housed in static caging with aspen bedding and the other groups, there was also an apparent difference in the uniformity of the cecal microbiota. As sample size calculations are partially based on the anticipated variance present in the outcome of interest, it is reasonable to believe that decreased variance in the composition of the GM through certain husbandry practices  could decrease the sample size needed to achieve adequate study power.Considering recent calls by the NIH for measures to enhance the reproducibility of preclinical researchIn conclusion, this study demonstrates that differences in factors such as caging and bedding can modulate the gut microbiota and that cecal samples might be optimal when screening for environmental effects on the gut microbiota. Ultimately, whether or not such changes in the composition of the microbiota have downstream effects on model phenotypes and associated study reproducibility will have to be determined on an individual basis but the possibility must be considered by researchers.N = 144) were purchased from Charles River Laboratories  in a single order, and housed in the same room and maintained under barrier conditions in microisolator cages on free-standing shelves or on individually ventilated cage-racks , filled with either compressed paper  or aspen chip bedding , with ad libitum access to autoclaved rodent chow  and acidified, autoclaved water, under a 14:10 light/dark cycle. Each treatment group representing a different combination of caging, bedding, and diet comprised three cages, and all cages contained a nestlet for enrichment and four mice per cage. Water was acidified using an automated bottle filler  designed to titrate municipal water with sulfuric acid to a target pH of 2.5 (range 2.3 to 2.7). Mice were determined to be free of all overt and opportunist bacterial pathogens including Citrobacter rodentium, Helicobacter spp., Mycoplasma spp., Pasteurella pneumotropica, Pseudomonas aeruginosa, Salmonella spp., Staphylococcus aureus; Encephalitozoon cuniculi; adventitious viruses including MHV, MVM, MS1 (generic parvo), MPV, MNV, TMEV, EDIM, Sendai, PVM, REO3, LCMV, Ectromelia, MAV1, MAV2, and polyoma viruses; intestinal protozoa including Spironucleus muris, Giardia muris, Entamoeba muris, Tritrichomonas muris, and other large intestinal flagellates and amoeba; intestinal parasites including pinworms and tapeworms; and external parasites including all species of lice and mites, via quarterly sentinel testing performed by IDEXX BioResearch . Mice were allowed to acclimate for a period of one week prior to the initial sample collection. At the end of study, mice were humanely euthanized via inhaled carbon dioxide, in accordance with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition, followed by cervical dislocation as a secondary means. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and under approval of the University of Missouri Institutional Animal Care and Use Committee.Four week-old female outbred Crl:CD1 (ICR) mice  weeks later, mice were humanely euthanized and jejunal, ileal, cecal, and fecal samples were collected using aseptic technique. Briefly, each region of the gut was exteriorized to allow collection of samples from roughly the same site of each animal. In addition to collecting luminal contents, the mucosa was gently scraped to ensure that mucosa-associated microbes were included in samples. Jejunal samples were collected from the approximate middle of the jejunum; ileal samples were collected from approximately 3 cm proximal to the ileocecocolic junction; cecal samples comprised the entire cecal contents; and fecal samples represented the most distal fecal bolus present in the GIT, excluding boli within the rectum proper. Instruments used for collection were flamed and allowed to cool between all samples. Following collection, all samples were immediately placed in 2 mL round-bottom tubes containing 800 µL lysis bufferFollowing mechanical disruption using a TissueLyser II , tubes were incubated at 70 °C for 20 minutes with periodic vortexing. Samples were then centrifuged at 5000 × g for five minutes at room temperature, and the supernatant transferred to a clean 1.5 mL Eppendorf tube. Two hundred µL of 10 mM ammonium acetate was added to lysates, mixed thoroughly, incubated on ice for five minutes, and then centrifuged as above. The supernatant was then aspirated, mixed thoroughly with one volume of chilled isopropanol, and incubated on ice for 30 minutes. Samples were then centrifuged at 16000 × g for 15 minutes at 4 °C. The supernatant was aspirated and discarded, and the DNA pellet washed several times with 70% ethanol and resuspended in 150 µL of Tris-EDTA. Fifteen µL of proteinase-K and 200 µL of Buffer AL  were added and samples were incubated at 70 °C for 10 minutes. Two hundred µL of 100% ethanol was added and the contents of each tube were transferred to a spin column from the DNeasy kit. DNA was then purified according to the manufacturer’s instructions and eluted in 200 µL of EB buffer (Qiagen). Yield was determined via fluorometry  using the quant-iT BR dsDNA reagent kit .38. Oligonucleotide sequences are available at proBase39. A single forward primer and reverse primers with a unique 12-base index were used in all reactions. PCR reactions (50 µL) contained 100 ng of genomic DNA, forward and reverse primers (0.2 µM each), dNTPs (200 µM each), and Phusion High-Fidelity DNA Polymerase (1U). PCR amplification was performed as follows: 98 °C(3:00) + [98 °C(0:15) + 50 °C(0:30) + 72 °C(0:30)] × 25 cycles + 72 °C(7:00). Amplified product (5 µL) from each reaction was combined and thoroughly mixed; pooled amplicons were purified by addition of Axygen AxyPrep MagPCR Clean-up beads to an equal volume of 50 µL of amplicons and incubated at room temperature for 15 minutes. Products were washed multiple times with 80% ethanol and the dried pellet resuspended in Qiagen EB Buffer (32.5 µL), incubated at room temperature for 2 minutes, and then placed on the magnetic stand for 5 minutes. The final amplicon pool was evaluated using the Advanced Analytical Fragment Analyzer automated electrophoresis system, quantified with the Qubit fluorometer using the quant-iT HS dsDNA reagent kit, and diluted according to Illumina’s standard protocol for sequencing on the MiSeq.Extracted DNA was processed at the University of Missouri DNA Core Facility. Bacterial 16S rDNA amplicons were constructed via amplification of the V4 hypervariable region of the 16S rDNA gene with universal primers (U515F/806 R), flanked by Illumina standard adapter sequences40, and culled if found to be short after trimming for a base quality less than 31. Qiime v1.841 software was used to perform de novo and reference-based chimera detection and removal, and remaining contiguous sequences were assigned to operational taxonomic units (OTUs) via de novo OTU clustering and a criterion of 97% nucleotide identity. Taxonomy was assigned to selected OTUs using BLAST42 against the Greengenes database43 of 16S rRNA sequences and taxonomy. Principal coordinate analyses, performed using ¼ root-transformed OTU relative abundance data, and alpha-diversity indices were determined using the Past 3.15 software package44. Heatmaps of log-transformed relative abundance data arranged via hierarchical clustering were generated using Metaboanalyst 3.046. The supplementary video was generated using unweighted UniFrac distances and the Qiime script beta_diversity_through_plots.py. Frames of data for animation were created using the R package rgl (r-forge.r-project.org/projects/rgl. ImageMagick (www.imagemagick.org) was used to create the animation.Assembly, binning, and annotation of DNA sequences was performed at the MU Informatics Research Core Facility. Briefly, contiguous DNA sequences were assembled using FLASH software17) of Bray-Curtis and Jaccard distances using Past 3.1544. Jaccard distances are an unweighted metric, based on the shared presence or absence of taxa between samples; Bray-Curtis distances  are weighted and thus factor in similarities in relative abundance of shared taxa. When making pairwise comparisons among all twelve groups, Bonferroni’s method was used to correct for multiple testing.Differences between groups in richness and alpha-diversity metrics were tested using a three-way analysis of variance (ANOVA) performed via a general linear model in SigmaPlot 13.0. Differences between groups in the relative abundance of 25 independently filtered operational taxonomic units with the highest loading scores were tested using a similar method although data were first quarter-root transformed to normalize for high sparsity. Differences between groups in beta-diversity were determined via one-way permutational multivariate analysis of variance  Sequence Read Archive (SRA) under BioProject accession number PRJNA382943.Supplementary Figures and TablesSupplementary Video S1.


Sauripes hadongensis, n. ichnogen., n. ichnosp., which represents the oldest lizard tracks in the world. Most tracks are pes tracks (N = 25) that are very small, average 22.29 mm long and 12.46 mm wide. The pes tracks show “typical” lizard morphology as having curved digit imprints that progressively increase in length from digits I to IV, a smaller digit V that is separated from the other digits by a large interdigital angle. The manus track is 19.18 mm long and 19.23 mm wide, and shows a different morphology from the pes. The predominant pes tracks, the long stride length of pes, narrow trackway width, digitigrade manus and pes prints, and anteriorly oriented long axis of the fourth pedal digit indicate that these trackways were made by lizards running bipedally, suggesting that bipedality was possible early in lizard evolution.Four heteropod lizard trackways discovered in the Hasandong Formation (Aptian-early Albian), South Korea assigned to  It is, in general, because their small bodies require a suitable depositional environment for preservation. Crown-group Squamata originated between the Late Triassic and the Early Jurassic (213~176 Ma) based on molecules and fossils2. Some skeletal materials of Iguania, Gekkota, Scincoidea, Lacertoidea, and Anguimorpha have been reported in Asia, Europe, and North America by the Early Cretaceous3. Unfortunately, fossil footprint records attributable to lizards are even rarer than those of body fossils because of the general light body weight of lizards and their preferred range of habitats4. There are thus far only three previous reports of fossil lizard tracks. Two were attributed to a lizard without a description from the Eocene Green River Formation, Utah: one is a lizard trackway6 and the other is one isolated track7. A new lizard trackway, Neosauroides koreaensis, was recently named from the Haman Formation (Late Albian~Early Cenomanian) of South Korea8.Although lizards are the most successful of modern reptiles in terms of the number of species  and their wide geographical distribution9, South Korea which are extraordinarily well-preserved and allow identification of detailed foot anatomy. Associated with a complete manus imprint, most tracks are very small (less than 26 mm) and appear as ectaxonic  and asymmetrical imprints which are frequently recognized as a diagnostic feature of the “typical” lizard pes10. Many modern lizards can bipedally run on the land and even on the surface of water 11. However, it has been unclear as to when lizards developed a capability for bipedal locomotion, though bipedal locomotion has been inferred in some fossil lizards based on the relationship between forelimb and hind limb lengths 13. Therefore, the discovery described in this report is highly significant because it is the first direct evidence of bipedal locomotion in fossil lizards, suggesting that lizard bipedality is deeply rooted in the phylogeny of lizard evolution.Here we describe the oldest crown-group lizard trackways known anywhere in the world which show bipedal locomotion. Tracks come from the Hasandong Formation (Aptian~early Albian)Order Squamata Oppel, 1811Sauripes hadongensis ichnogen. et ichnosp. nov.Ichnogenus named from ancient Greek “sauros” (lizard) and “pes” (foot). Ichnospecies named after Hadong County that yielded the holotype.Manus and pes prints on a mudstone slab (70 × 30 cm) .9, an abandoned quarry next to Hadong power plant, Hadong County, South Gyeongsang Province, South Korea Quadrupedal tracks; manus prints are medial to the pes prints; the pes prints are larger than the manus prints; plantigrade and pentadactyl pes prints are longer than wide; the digit length progressively increasing from digits I to IV (ectaxonic); digit V is oriented more laterally and offset from other digits; digit imprint IV is more than twice the length of the metatarsal impression; plantigrade and pentadactyl manus print has similar length and width dimensions; digits II and IV are shorter than digit III (mesaxonic); the interdigital angle between digits I and V of the manus is larger than that of the pes.The slab contains 29 lizard tracks without any other vertebrate traces Fig. . They arBased on track morphology, two different types of tracks are observed on this slab. One type  has curved digit imprints that progressively increase in length from digits I to IV, a smaller digit V that is distinctly separated from the other digits by a large interdigital angle, and digit V is oriented more laterally. The other type  is mesaxonic, having a longer digit III compared to the others . The average manus and pes length is 19.18 mm and 22.29 mm, respectively (heteropody). Although manus-pes sets are not regularly imprinted, the tracks visible on the slab clearly show locomotion patterns without tail trails.Of the four manus tracks, one in trackway B B1, Fig.  is betteThe pes prints are plantigrade, pentadactyl, and distinctly ectaxonic Figs , 3. Digi14 and varanids15. Trackway A is the longest and best preserved among the four trackways, comprising one incomplete right manus and nine pes prints (4 left and 5 right). The average pes stride length is 79.18 mm and the average pace length is 47.82 mm with 112.88° as pace angulation. The trackway width becomes narrower as stride length increases in trackways A and B  are recognized for locomotion in lizards18. Many lizards (more than 50 species) are known to have the capability for bipedal locomotion19. Although some lizards appear to run bipedally without acceleration19, bipedality usually occurs as a consequence of acceleration in a lizard with hind limbs that are significantly longer than the forelimbs, moving the center of mass, and the rotational force on the hip joints21. When lizards are moving at relatively slow speeds, they retain a sprawling limb posture with laterally oriented plantigrade feet22. With this locomotor pattern, the front feet are positioned under the body with the head up, increasing the chance of leaving manus imprints rather than pes ones as seen in Neosauroides koreanesis8. In contrast, Sauripes hadongensis shows pes-dominant trackways characterized by long strides, a large pace angulation, and digitigrade prints throughout the locomotion sequences. Snyder pointed out that long hind limbs, short forelimbs, a narrow pelvis and a long tail could aid bipedality in lizards, mostly through increased stride length23. Running lizards at high speeds frequently also leave digitigrade footprints rather than plantigrade ones24.Many extant lizard species can run bipedally, but not as obligate bipedality. Lizards exhibit different gaits, from quadrupedal and bipedal species to terrestrial and arboreal specialists. Lizard locomotion is strongly influenced by body shape and length, as well as by differences in habitatS. hadongensis has better defined impressions of the digits than of the sole pads, with distinctly deep metatarsophalangeal joint in digits II, III, and IV, behind which the sediment is slightly pushed up, indicating that they ran mainly on the digits, instead of touching the whole soles plantar surface on the substrate  are preserved before the transition to bipedal locomotion. The lizard is the only vertebrate animal that starts on all limb pairs, then transitions to the bipedal gait by acceleration17. Bipedality in lizards may be advantageous for enhanced environmental perception during locomotion by elevating the head and expanding the visual field during obstacle negotiation28. It is not certain whether S. hadongensis tracks were made when escaping from predators or not, but interestingly, the pterosaur track Pteraichnus koreanensis was reported from the same horizon at the same site29. Some pterosaurs likely foraged in diverse environments for small animals and carrion30. The occurrence of P. koreanensis and S. hadongensis tracks together may imply that these two trackmakers had a contemporary antagonistic relationship. If true, the threat of pterosaur predation might have caused these running lizards to leave the bipedal trackways found in the Hasandong Formation  already showed a capability for scansorial locomotion32. The Teiidae is native to the Americas34. In Asia, they first appeared in the Late Cretaceous in Mongolia and China35. Varanid lizards radiated from Mongolia during the Late Cretaceous to Early Cenozoic (80~50 Ma) and dispersed to almost all major fragments of Laurasia and Gondwana36.Bipedality can be observed in phylogenetically diverse extant lizard families such as Lacertoidea (teiids), Anguimorpha , and Iguania , particularly among the species that live in sandy, rocky or open environments39, primitive iguanians (acrodontans and non-acrodontans) existed in Laurasia by the Aptian/Albian. Extant iguanians usually have well-developed, strong legs suitable for bipedality21. In addition, the extinct polyglyphanodonts are known from the Early Cretaceous in Asia40 and became abundant in the Upper Cretaceous of Mongolia and China41. They have strong hind limbs and a rather iguanian skeletal morphology. Therefore, based on the palaeobiogeographic distribution of facultative extant families, the lizard that produced S. hadongensis tracks could well have been a member of an extinct family or stem members of Iguania, which was present in the Early Cretaceous.Based on fossils and molecular data9. The overlying Jinju Formation and underlying Nakdong Formation have been dated to 109.9 ± 3.2 Ma and 127.67 ± 1.3 Ma, respectively43. The Hasandong Formation has yielded the most abundant vertebrate body fossils in the Gyeongsang Supergroup (Barremian~Campanian), part of the largest Mesozoic Gyeongsang Basin in the Korean Peninsula. Vertebrate fossils include turtles, pterosaurs, crocodilians, and dinosaurs44. Most bones occur as scattered, broken, and isolated pieces which had probably undergone long aerial exposure, transportation, and scattering on the floodplain before burial45. Previously described vertebrate ichnofossils from the Hasandong Formation include dinosaur tracks46 and pterosaur tracks of Pteraichnus koreanensis29.The Hasandong Formation is inferred to be of Aptian to early Albian age based on a comprehensive paleomagnetic and radiometric data2 of exposure, representing the middle part of the Hasandong Formation , presenting sediments thought to have been deposited in small swamps and/or marginal lakes associated with floodplains between channels.The lizard track site is from an abandoned quarry next to the Hadong power plant, Hadong County where there is approximately 5,000 mSupplementary InformationSupplementary Information Table 1


Functional and morphological evolution of remnant pancreas after resection for pancreatic adenocarcinoma is investigated.The medical records of 45 patients who had undergone radical resection for pancreatic adenocarcinoma from March 2010 to September 2013 were reviewed retrospectively. There were 34 patients in the pancreaticoduodenectomy (PD) group and 10 patients in the distal pancreatectomy (DP) group. One patient received total pancreatectomy. The endocrine function was measured using the glucose tolerance index (GTI), which was derived by dividing daily maximum serum glucose fluctuation by daily minimum glucose. Remnant pancreas volume (RPV) was estimated by considering pancreas body and tail as a column, and head as an ellipsoid, respectively. The pancreatic atrophic index (PAI) was defined as the ratio of pancreatic duct width to total pancreas width. Representative indices of each patient were compared before and after resection up to 2 years postoperatively.P < .001). Overall, GTI increased on postoperative day 1 , and then decreased by day 7 . In the PD group, the GTI on POD#14 became lower than preoperative . PAI in the PD group was significantly lower at 1 month postoperatively . In the PD group, RPV was significantly lower at 1 month postoperatively , due to the resolution of pancreatic duct dilatation. RPV of the DP group showed no significant change. GTI was negatively related to RPV preoperatively , but this correlation disappeared postoperatively .The area under receiver operating characteristic curve of GTI for diagnosing DM was 0.823 (95% confidence interval, 0.699–0.948, Pancreatic endocrine functional deterioration in pancreatic adenocarcinoma patients may in part be due to pancreatic duct obstruction and dilatation caused by the tumor. After resection, this proportion of endocrine insufficiency is corrected. Although ideally as much pancreatic parenchyma should be preserved as possible to maintain good endocrine and exocrine pancreatic functions, sometimes only a small volume of pancreas is left after radical resection, and concerns regarding pancreatic insufficiency develop especially when a tumor is accompanied by parenchymal atrophy.–6 Most studies published so far on the functional and morphological evolution of remnant pancreas after resection had been conducted on patients with diverse disease entities that differ in the degrees of pancreatic duct dilatation,,7–11 which can adversely affect pancreas function. Furthermore, the method usually used to evaluate pancreas endocrine function was developed for diabetes mellitus (DM), without consideration of the lower levels of blood glucose governed by glucagon, an important counter-regulatory hormone in glucose metabolism. The aim of this study was to examine the functional and morphological evolution of remnant pancreas after resection for pancreatic adenocarcinoma by developing a model based on readily available clinical data. We tried to devise a new index for pancreatic endocrine functional assessment and evaluated morphological changes of remnant pancreas by approximating it to simple geometric solid figures in accord with appearances on abdominal computed tomography (CT) images. The correlations between these parameters and their evolutions after surgical resection were analyzed and the implications were sought.Surgical removal provides the best survival benefit for patients with pancreatic cancer.22.1From March 2010 to September 2013, a total of 46 consecutive patients with pancreatic adenocarcinoma underwent pancreatic resection with radical intent at our institute, and of these, 45 were included in this study—the excluded patient developed pancreatic cancer from an intraductal papillary mucinous neoplasm. In addition, 1 patient that had undergone total pancreatectomy was excluded from the evolutional analysis. Medical records and CT images of the enrolled patients were obtained and reviewed retrospectively. Data were collected preoperatively and until the first detection of recurrence, distant metastasis, death, or 2 years after the operation. The patients were divided into 2 groups, the pancreaticoduodenectomy group  and the distal pancreatectomy with splenectomy group . The PD group was divided into 2 subgroups according to pancreaticoenteric anastomotic methods; the pancreaticojejunostomy subgroup  and the pancreaticogastrostomy subgroup , irrespective of pylorus preservation. In general, PG was performed when the pancreatic head cancer had been extended to the left of superior mesenteric vein, rendering conventional PJ technically difficult. The authors have decided that this study can be considered exempt from the institutional review board oversight, because this research collected existing data and the individual subjects cannot be identified directly.2.2 Using these criteria, patients were classified based on the preoperative presence or absence of DM. In patients with unstable serum glucose (SG) or with a preoperative history of DM, SG was checked 4 to 6 times daily, preoperatively and postoperatively until SG had stabilized after resumption of oral intake. Based on the assumption that pancreatic endocrine function is reflected by daily serum glucose fluctuation, we defined related parameters as follows:SGmax: Daily maximum serum glucose level, mg/dLSGmin: Daily minimum serum glucose level, mg/dLΔSG: Daily variation in SG, defined as SGmax – SGmin. In patients whose SG was stable enough to warrant no more than 1 sampling, ΔSG (mg/dL) was defined as the difference between sampled SG and 90 mg/dL.Ireq: Daily requirements of exogenous insulin equivalents administered to maintain SG within normal ranges, irrespective of resulting SG, as measured in the international unit (IU).Glucose tolerance index (GTI) = ΔSG/SGmin.DM was defined according to the criteria suggested by the American Diabetes Association.2.3The functional evolution of pancreatic endocrine function was estimated using GTI trends. Patient GTIs were checked preoperatively and at 1, 7, 14 days, and 1, 2, 3, 6, 12, 18, 24 months after the operation. When GTI values were not available for specific times, values the nearest to the study points were allocated.2.4 When a tumor occupied the area, PAI was measured at the nearest pancreatic parenchyma possible. In the DP group, PAI could not be measured because the main pancreatic duct within the remaining pancreas head was not dilated. Antero-posterior diameter (APD) and latero-lateral length (LLL) were measured from CT images in the axial view RPV of the pancreas head = 4π/3 × APD/2 × CCD/2 × LLL/2 (cm3)RPV of the pancreas body and tail = π × APD/2 × CCD/2 × LLL (cm2} = RPV × {1−(PAI)2} (cm3)Corrected RPV (cRPV) = pancreas parenchymal volume = RPV – main pancreatic duct volume  = RPV × {1 – (PDW/TPW)Morphological evolution was investigated using 2 indices, pancreatic atrophic index (PAI) and remnant pancreatic volume (RPV). In the PD group, PAI was defined as the pancreatic duct width (PDW) divided by total pancreatic width (TPW), as measured in the CT axial view anterior to the aorta Fig. A.16] Wh6 Wh WhenIn the DP group, the main pancreatic duct volume of the remnant pancreas head was neglected, and cRPV was assumed to be identical to RPV. The PAI and RPV were measured preoperatively, at 1, 3, 6 months postoperatively, and 6 monthly up to 2 years. All measurements were performed using the m-view 5.4 software .2.5U test was used to compare continuous variables, and chi-square test or Fisher's exact test was used to analyze categorical variables. The relevance of GTI with respect to the presence of preoperative DM was validated by receiver operating characteristic (ROC) curve analysis. Wilcoxon's signed rank test was used for intragroup evolutional analysis. Correlation between GTI and RPV was analyzed using Pearson correlation coefficients (r). Missing data were handled by listwise deletion. The analyses were performed using IBM SPSS Statistics for Windows, Version 19.0 , and a P value < .05 was considered statistically significant.Descriptive data were presented using median and range for continuous variables, and number and percent for categorical variables. The Mann–Whitney 33.1 One patient (2.9%) in the PD group died of bleeding related to grade C pancreatic fistula, and overall operative mortality was 2.2%. One patient (2.2%) had T1N1 pancreatic adenocarcinoma according to the American Joint Committee on Cancer staging system, 11 patients (24.4%) had T3N0, and 33 patients (73.3%) had T3N1 disease. None of the demographic characteristics differed between the PD and DP groups. Table Demographic data are presented in Table  patient .9% in th3.2P < .001; Fig. Preoperative SGmax, SGmin, ΔSG, and Ireq were significantly different in patients with or without DM Table . Of the 3.3One patient (2.2%) in the PD group showed overt steatorrhea during the immediate postoperative period, which improved on pancreatic enzyme supplementation. Two other patients in the PD group (5.9%) and 1 in the DP group (10%) complained of frequent greasy loose stools postoperatively. These conditions also subsided during follow-up after medication.P = .001; vs baseline), and then decreased to its preoperative value by POD#7 . In the DP group, GTI did not change significantly thereafter , and remained relatively unchanged thereafter .Preoperative (Baseline) GTI of the 44 study subjects was 1.02 ± 1.41 . On the first postoperative day (POD#1), GTI increased significantly  at 1 month postoperatively, and remained stable thereafter . In both groups, RPV remained relatively unchanged thereafter (P > .05).Preoperative PAI in the PD group was 0.38 ± 0.18, and this decreased to 0.22 ± 0.12  .Preoperatively, a moderate negative linear correlation was observed between GTI and RPV , or directly measured serum insulin levels.,20 However, these parameters are not checked routinely, involve additional cost, and more importantly, focus on insulin and its activity, which represent only 1 of the 2 arms of serum glucose regulation. To assess pancreatic endocrine function properly, the effect of glucagon, a counter regulatory hormone of insulin, must also be considered.,8,13 The action of glucagon can be considered a mirror image of that of insulin. Glucagon controls lower levels of serum glucose, as evidenced by the exacerbations of daily glucose fluctuations after total pancreatectomy (pancreatogenic DM), as compared to the diabetic patients in whom the glucagon secretion is preserved.,22,23 We assumed that the end-result of the complex glucose control networks were daily serum glucose fluctuations, which were quantified using ΔSG in the present study. By dividing ΔSG by the daily minimum glucose level (the definition of GTI), we tried to enhance ΔSG and differentiate patients with identical ΔSG values but different glucagon activity. Another important factor of serum glucose regulation is exogenous hypoglycemic agents. However, it has been reported that exogenous insulin supplementation and oral hypoglycemic medications affect serum glucose usually a 1-sided manner and would not significantly alter ΔSG were it not for counter regulatory hormones. Of note, a lowering of GTI does not necessarily mean that blood glucose has decreased. Rather, it should be interpreted as an indicator of improved glucose control, and although DM may develop or worsen postoperatively, GTI reduction indicates the condition is easier to control, presumably through the preserved glucagon function.Usually pancreas endocrine insufficiency is diagnosed based on the presence of DM. However, the procedure is time-consuming and requires additional software. Djuric-Stefanovic et al proposed a simpler method for measuring the pancreatic volume by treating the pancreatic head as a column and the body and tail as a square pillar. In the present study, we likened the pancreatic head to an ellipsoid and the body and the tail to a cylinder. Our method tended to underestimate actual pancreas volumes, but calculated dimensions were comparable to previous reports,,25 except pancreas body length, which was shorter because we measured the length perpendicular to the resection plane. Recently, Yoo et al reported the evolution of RPV during the 12 months following PD in patients with diverse periampullary tumors, and compared RPVs between patients after external and internal drainage. Although they did not mention the degree of pancreatic duct dilatation, their results showed that after PD, RPV values decreased progressively with time presumably due to pancreatic atrophy. However, it is unclear whether this decrease in RPV values was due to the resolution of pancreatic duct dilatation or the wasting of remnant parenchyma. Lemaire et al reported that in their series of 17 patients who had undergone PD and PG, pancreatic parenchymal thicknesses decreased and main pancreatic ducts dilated significantly after surgery. Their findings regarding main pancreatic duct evolution contradict ours possibly because of the method of pancreaticoenteric anastomosis, PG, which current study involves only 5 cases. However, they did not include pancreatic adenocarcinoma, and thus preoperative pancreatic duct dilatation was not likely. Also, Kim et al showed that pancreatic duct sizes progressively increased in the invagination group but not in the duct-to-mucosa group after PD. In view of the facts that they used a duct-to-mucosa technique in patients with duct dilated to >3 mm in diameter, and that their data included 24 (17%) patients with pancreatic carcinoma, it is probable that postoperative pancreatic duct dilatation in their series was limited to those with periampullary diseases other than cancer of the pancreatic head. The present study shows that the reduction in RPV after PD was due to a decrease in PAI, and hence, to a decrease in pancreatic duct volume, as was confirmed by the relatively unchanged corrected RPV values of patients with adenocarcinoma of the pancreatic head. As for the morphologic evolution in a DP group, Phillip et al reported serial increases in pancreatic head volumes in their series of 24 patients. They suggested that increases in pancreatic head volume were due to regeneration, which mandates further investigations be undertaken to differentiate hypertrophy and simple swelling. In the present study, the DP group showed the same increasing trend in pancreas head volume, but too few cases were enrolled to achieve statistical significance.Pancreatic volume can be measured by CT volumetry.,5,20 although there are contradictory reports. In general, an RPV of 20% is considered the minimum requirement to avoid clinically relevant DM, provided that pancreas parenchyma is free of diffuse pathology after PD or DP. Recently, Burkhart et al reported 48% of patients experienced aggravation of preexisting DM after PD as compared with 26% after DP, and that 18% of patients developed DM after PD as compared with 31% after DP. This seemingly disproportional aggravation of DM in patients after PD and DP could be due to the different distributions of pancreatic α- and β-cells, which secrete glucagon and insulin, respectively. When we studied the evolution of pancreas endocrine function using GTI instead of DM, GTI was observed to increase significantly during the immediate postoperative period, which probably reflects acute glucose tolerance deterioration due to operation, inflammation, and infection. At 7 days postoperatively, when most patients resumed oral intake, indulged in active exercise, and systemic inflammation was under control, GTI decreased significantly, and subsequently remained stable. The favorable GTI profile after the immediate postoperative period was observed mainly in the PD group, presumably because more pancreas parenchyma is preserved after PD and more α-cells remain. However, these explanations inadequately explain why GTI improved versus preoperative values after pancreatic resection, which inevitably involves loss of pancreatic volume. It could be that after PD, pancreatic duct obstruction caused by pancreatic cancer is relieved, and thus remaining distal pancreas is decompressed. Pancreatic adenocarcinoma is frequently accompanied by atrophy and duct dilatation distal to the tumor, and duct obstruction can interfere with pancreatic endocrine function and can cause DM.,14 There are reports that preoperative DM improved after PD in some patients,,12,30 supporting our data that the acute pancreatic duct dilatation caused by obstruction by pancreatic cancer is reversible. Another mechanical change that accompanies is the removal of tumor mass, which can secrete diabetogenic agent. However, we could not observe the improvement of GTI after DP, although the tumors had been removed in both PD and DP groups. The causal relationship between pancreatic cancer and DM is unclear, although some evidence indicates DM caused by pancreatic cancer may be reversible after surgical resection. In the present study, the prevalence of preoperative DM in all patients was 53.3%, which was more than twice as higher than values reported on mixed disease entities.,10,12 Considering that baseline serum glucose was measured during the immediate preoperative period, it is likely that so-called acute or new-onset DM cases caused by pancreatic cancer, whether by duct obstruction or tumor-related diabetogenic material, were included in the present study.,30 Our results show that such cases may exhibit improved pancreas endocrine function after removal of the tumor burden and amelioration of duct obstruction by surgical resection, as evidenced by the evolution of GTI and PAI, and the disappearance of the correlation between GTI and RPV postoperatively. DM is the hallmark of pancreatic endocrine insufficiency, and is a multifactorial entity that embraces volume-related factors, such as insulin and glucagon secreting abilities, and cancer-related factors, such as tumor burden and duct obstruction and dilatation. The present study shows the latter can be corrected by surgical resection, at the cost of pancreas volume. Surgery for pancreatic adenocarcinoma should be approached differently from other diseases requiring pancreas resection, in terms of remnant pancreas volume and the risk of developing postoperative endocrine insufficiency, because DM arising from pancreatic adenocarcinoma is different.Pancreatic resection has been reported to predispose new-onset DM and worsen glucose control after partial or total pancreatectomy and that the percentage of pancreatic parenchymal loss is roughly correlated with postoperative endocrine insufficiency, as evidenced by newly developed DM and worsening of glucose intolerance,The major limitation of this study comes from the design itself, which is retrospective in nature. Clinical data were sometimes missing at certain study points, due to individual patient's follow-up schedule not being standardized as well as poor prognosis of pancreatic adenocarcinoma. A limited number of relevant cases made parametric analysis not applicable, decreasing the power of statistical analyses. Further studies with better-designed protocol and more cases are warranted. Comparison between pancreatic head cancers with distal pancreatic duct obstruction and other periampullary cancers without pancreatic duct dilatation can further clarify the results of this study. Also, GTI should be refined to evaluate pancreatic endocrine function more accurately, including other related variables such as oral hypoglycemic agents and exogenous insulin supplements.In conclusion, the pancreatic endocrine function can be evaluated using the glucose tolerance index, which is defined as the ratio of daily serum glucose fluctuation and daily minimum glucose level, and thus, is not based wholly on the status of diabetes mellitus. In patients with pancreatic adenocarcinoma, the pancreatic endocrine function is further compromised by pancreatic duct obstruction and dilatation caused by the tumor, and this proportion of pancreatic endocrine dysfunction can be reversed by standard pancreatic resection.


Variation in PA effectiveness is largely driven by local factors affecting individual PAs, rather than designations assigned by governments.Tropical deforestation is responsible for around one tenth of total anthropogenic carbon emissions, and tropical protected areas (PAs) that reduce deforestation can therefore play an important role in mitigating climate change and protecting biodiversity and ecosystem services. While the effectiveness of PAs in reducing deforestation has been estimated, the impact on global carbon emissions remains unquantified. Here we show that tropical PAs overall reduced deforestation carbon emissions by 4.88 Pg, or around 29%, between 2000 and 2012, when compared to expected rates of deforestation controlling for spatial variation in deforestation pressure. The largest contribution was from the tropical Americas (368.8 TgC y Carbon sequestration and storage in terrestrial systems is an important mechanism for climate change mitigation3, and deforestation and land clearing are significant drivers of carbon emissions in tropical systems, as deforestation is the most important contributor to emissions from land use change4. Between 2000 and 2010, tropical deforestation and land use change emitted 1.0 PgC y−1 net to the atmosphere6. When forest degradation is also included7, tropical emissions between 1990 and 2010 were around 1.4 PgC y−1.Tropical forests account for around 68% of global forest carbon stocks, in terms of live biomass, above and below ground10, and are intended to protect habitats11 as well as benefit local communities through ecosystem service provision and the support of sustainable livelihoods12. Although PAs can achieve conservation goals14, deforestation does occur within PAs9; in some cases increasing human pressure (hunting and timber extraction nearby) is driving biodiversity loss and decline11. While it may be difficult to achieve both biodiversity and socio-economic benefits, there is evidence that where people are included as stakeholders, mutually beneficial scenarios are achievable15. PAs vary widely in extent and type, from local ecological or indigenous reserves through to national parks and World Heritage Sites. The International Union for Conservation of Nature (IUCN) categorization system comprises six broad classes, relating to their management objectives17: Category Ia denotes strict nature reserves, Ib wilderness areas without significant human habitation, II national parks set aside to control large-scale ecological processes, III natural monuments or feature likely to have high visitor value, IV habitats or species management areas often requiring management intervention, V protected landscapes or seascapes featuring significant human influence, and VI areas with sustainable use of natural resources. The ability of category V and VI to both conserve biodiversity and promote economic welfare remains contested17.Protected areas (PAs) comprise up to 15% of the land surface globally across all ecosystems19, but there has been little analysis of the impacts on carbon stocks and losses21, which influence climate change-related biophysical feedbacks22. While total soil carbon content in forests is 2-3 times greater than that stored in above ground biomass (AGB)5, it is not always readily released to the atmosphere after deforestation; whether it is or not depends on the post-deforestation land use5. Root, or belowground, biomass (BGB) is generally equivalent to one-fifth of the AGB in tropical forests23: here we focus on AGB, in line with other recent studies3. Recent high-resolution forest cover24 and above ground biomass (AGB)5 estimates allow large-scale analysis of PAs in terms of reduction of forest canopy loss and carbon emissions. We analysed the effectiveness of PAs in reducing deforestation and forest carbon loss, investigating variation among PA designations and controlling for spatial variation in deforestation pressure. Using empirical relationships between canopy cover and AGB, we estimated the influence of PAs on tropical forest carbon emissions resulting from reductions in deforestation rates.Previous tropical or global analyses of the effectiveness of PAs have focused on biodiversity conservation, forest loss or social outcomes26, and they are especially important for forest conservation in developing countries27. However, the siting of PAs is not random, and they are often located in areas that are inaccessible or unsuitable for agriculture, far from cities or transport links, and in topographically challenging areas , such that they are unlikely to be under pressure from the developmental drivers of land use change8. Because of non-random PA locations, and because deforestation rate varies among countries due to political and socioeconomic factors9, PA effectiveness may be overestimated28, and so statistical models of forest loss in unprotected (non-PA) areas are commonly used to control for this bias29. We analysed the difference (rd) between observed and expected remaining forest cover in PAs to control for any biases in PA location. The expected remaining cover was determined from Generalized Additive Models (GAMs) of forest loss in non-PA regions. Positive rd therefore indicates that remaining forest cover in PAs is greater than expected for a particular location, and negative rd indicates that remaining forest cover is less than expected.Deforestation rates within PA borders are significantly lower than outsideWe analysed the three major tropical regions, the Americas, Africa, and Asia, separately , 0.39 ± 0.10% for Africa , and 1.92 ± 0.17% for Asia   for the Americas, 0.004 ± 0.026%  for Africa, and 0.027 ± 0.061%  for Asia, confirming unbiased estimation of deforestation by the GAMs. PAs in the Americas have positive rd across the range of predicted remaining canopy cover, while the African and Asian PAs are most effective at 60–80% of predicted remaining cover  21 included reduction of deforestation as a key activity in mitigating climate change to 2020, and our analysis suggests that tropical PAs have played a significant role in conserving forest carbon in recent years, equivalent to a reduction of around 29% of tropical deforestation emissions. Global carbon sinks will be critical for meeting the Paris Agreement, and tropical deforestation risks rainforest carbon sinks switching to carbon sourcesrobs) in all forest areas, using data on forest cover in 2000 and loss by 2012. Second, we constructed statistical models of robs in unprotected areas using a number of land-use and environmental predictors. Third, we predicted the expected remaining canopy cover (rexp) in PAs, from predictor variables within PAs using the models. Fourth, we calculated the difference between observed and expected canopy cover as rd = robs−rexp, to give a measure of PA effectiveness relative to background deforestation pressure. Fifth, we estimated empirical relationships between above-ground forest carbon density and canopy cover, and used these relationships to estimate the difference between observed and expected remaining carbon within PAs.In summary, our analysis proceeded in five stages: first, we estimated observed remaining canopy cover in 2012  was used to analyse the effectiveness of PAs in reducing deforestation. Positive rd values indicated that remaining cover was greater than expected for a non-PA area with the same values of predictor variables, and negative rd indicated that remaining cover was lower than expected. GAMs have been previously used in analyses of PA effectiveness29, and are useful when the underlying relationship between predictor and response is non-linear, and is not readily defined by a particular mathematical function. We checked for bias in GAM estimates of remaining forest cover for non-PA areas by testing rd vs. zero, using spatial block bootstrap resampling37 with data aggregated to 1° blocks, weighted by the fraction of non-PA forest pixels per block, and 1000 bootstrap samples. All analyses were conducted in R v.3.4.0.ref.38.Full descriptions of all datasets are given in the Supplementary Information. We estimated the mean remaining canopy cover from original canopy cover in 2000 and loss by 2012, by setting those pixels reported as having lost forest to zero canopy, under the assumption that forest loss was complete for those pixels, and that forest would not have grown back by 2012. The original data were at 1 arc second (~30 m) resolution, and we aggregated these to 1 arc minute (~1.8 km) resolution by taking means. We used Generalized Additive Models (GAMs)29: Country (as a fixed factor), human population density39, road density40, agricultural suitability41, altitude42 and fraction of steep slopes42 as the area per pixel with a slope >15°. All continuous variables were fitted as cubic regression splines , and we included spatial location as a two-dimensional tensor product smoothed across latitude and longitude to control for other, unidentified sources of spatial variation in deforestation pressure. No other interaction terms were included, and separate models were fitted for each tropical region . All datasets were transformed to 1 arc minute, either by aggregation to the mean value or bilinear interpolation, if the original dataset was at higher or lower resolution, respectively. The responses of remaining cover to predictors are shown in Supplementary Fig. In addition to original canopy cover in 2000, we used the following predictors of remaining forest cover in 2012 in our GAMs, which have all been implicated in previous studies of tropical deforestationThe difference between predictions from GAMs including all our deforestation pressure proxies  and null model using only original canopy cover was small indicating a relatively small influence of these predictors on deforestation pressure Fig. . A fractrd) was calculated for each PA, omitting PAs with total areas below the minimum pixel size of 1 arc minute (approximately 300 ha). PAs above this threshold represented 97%, 94% and 90% of the total PA area in the Americas, Africa and Asia, respectively. We then fitted GAMs to PA mean rd weighted by PA area, such that larger PAs were more influential on the fit . To estimate biomass in 2000 and 2012 we derived models of biomass from canopy cover and ecosystem type, for only those pixels that did not change canopy cover by more than 1% between 2000 and 2012. We aggregated biomass to 1 arc minute resolution, and fitted a smoothing spline using canopy cover as the predictor for each tropical region , using GAMs. Inclusion of ecosystem type improved the model R2 from 92.7 to 93.4% for the Americas, from 90.0 to 90.5% for Africa, and from 90.8 to 91.8% for Asia. These improvements in model fit were trivial and so we estimated original and remaining biomass from original and remaining canopy cover using the simple models, omitting the minor variations due to ecosystem class. We then estimated AGB from the predicted remaining canopy cover for PAs, using the remaining canopy cover model parameterized using non-PA areas. AGB per unit area was converted to above-ground carbon by multiplying the AGB by pixel area in hectares, and using a factor of 0.5 to convert from biomass to carbon45. The difference between the remaining AGB estimates and the predicted remaining AGB estimates for pixels within PAs was summed to give the overall carbon saving per region. Only pixels with original canopy cover greater than 20%, and within the tropics (between 23°N and 23°S) were included in the carbon saving calculation.We estimated carbon loss from deforestation using 15 arc second resolution aboveground live woody biomass (AGB) data32. We tested for an effect of local leakage by re-fitting GAMs for remaining forest cover in 2012, but excluding non-PA areas in a buffer around all PAs20. We chose buffer widths of similar magnitude to those used in a previous study20, but which increased with PA size such that buffer depth in kilometers equalled the natural logarithm of PA area in square kilometres (truncated to zero below 1 km2 area), as the potential for displacement of deforestation range of 5 km for the majority of PAs, used in the previous study, with a maximum around 10 km for the largest PAs (Supplementary Fig. 20.Leakage, the displacement of deforestation from inside to outside PAs, could bias the results by spuriously elevating the observed deforestation rate in non-PA regionsSupplementary Information


Escherichia coli (E. coli) and turbidity, were used to assess the water quality of 97 wells. Additionally, the study looked at the microflora diversity of the water, focusing on potential pathogens using outgrowth, PCR, and genome sequencing for 10 wells. Concentrations of TC for the open dug wells (4 × 104 CFU/100 mL) were higher than the drilled (2 × 103 CFU/100 mL) and closed dug wells (3 × 103 CFU/100 mL). E. coli concentration for drilled and closed dug wells was <22 MPN (most probable number)/100 mL, but higher for open wells (>154 MPN/100 mL). The drilled well turbidity (11 NTU) was within the standard deviation of the closed well (28 NTU) compared to open dug wells (49 NTU). Drilled and closed wells had similar microbial diversity. There were no significant differences between drilled and closed dug wells. The covering and lining of hand-dug wells should be considered as an alternative to improve access to safe and clean water in rural communities.The objective of this study was to examine three well designs: drilled wells (20–30 m deep), closed dug wells (>5 m deep), and hand-dug open wells (<5 m deep), to determine the water quality for improving access to safe and clean water in rural communities. Heterotrophic plate count (HPC), total coliforms (TC),  Sub-Saharan Africa is inhabited by a total of 783 million people, 40% of which do not have access to safe and clean water . The reg2 with a total population of 407,880 [In 2010, a non-profit organization, Maji Safi kwa Afya Bora Ifakara , began a project to improve rural water sources in the Kilombero region by drilling boreholes 20–30 m deep. The study focused on three wards within the Kilombero region in Tanzania; Ifakara, Namawala and Idete . The reg 407,880 . The pop 407,880 . Data foTo improve access to safe and clean water, MSABI began installing drilled rope pumps. These are lined and covered boreholes. They also started another project to modify hand-dug open wells into closed wells by providing lining, installing a locally made rope pump and covering the top of the well. Prior to MSABI’s efforts, it was estimated that the region had over 750 water points with more than 75% being shallow dug wells and surface water . By 2011This study compared three types of water sources: deeper drilled boreholes (20–30 m), hand-dug wells converted into closed wells (depth varies but between 5 and 10 m), and the shallow hand-dug wells (depth < 5 m). The hand-dug wells with depth > 5 m are internally lined and the tops are covered. These two improved well designs (the borehole and the open top converted into a closed well) will be referred to as MSABI rope pump well, and closed well, respectively. Improved water sources as defined by the Joint Monitoring Programme (JMP) are sources that, by their very nature of construction, adequately protect the source from outside contamination in particular with fecal matter . ExampleThe research question in this study was to examine the benefits of deeper, or shallow protected wells, and whether deeper wells offer water of better quality than shallow wells. The findings of this study will provide further guidance on access and water supply development in Tanzania and Sub-Saharan Africa as a whole. A total of 97 samples were collected with an average of 32 wells from each village: Ifakara, Namawala, and Idete. Approximately 11 of each of the three kinds of wells  were sampled from each village. Due to limited resources, only 10 wells from Ifakara village were analyzed for microflora focused on potential pathogens. Three of each well design were analyzed. The types of wells and depths are included in E. coli was determined using Colilert Test Kits . E. coli is a fecal coliform, or bacteria, whose presence indicates fecal contamination from human or animal waste [E. coli assessment using the Colilert test. Water samples were collected using plastic sterile whirl-packs and transported to the Ifakara Health Institute (IHI) laboratory using an ice chest. The sampling took place from early morning until late afternoon and the water samples were processed within 8 h of collection. For heterotrophic plate counts and total coliforms, 1 mL of each sample was cultured onto a 3 M Heterotrophic Count Plate (AQHC) Petrifilm  via spread plating after dilution in phosphate-buffered saline . Heterotrophs, which require organic carbon for growth, were plate counted for well comparison purposes only. Coliforms are a group of bacteria which are aerobic and facultative anaerobic, Gram-negative, non-spore formers, rod-shaped, and ferment lactose with the formation of gas within 48 h at 35 °C. Total coliforms are naturally present in the environment. Even though total coliforms are not useful as indicators of fecal pathogens, they are, however, useful indicators of other pathogens in drinking water . For undal waste . The tesIn addition to the detection of microbial water quality indicators, microflora diversity was also of interest for comparison purposes. However, due to limitations of the types of analysis that can be done in remote areas, water samples were collected and shipped to the United State via express delivery, which were then collected by a trained MSABI technician. The water samples were collected in 15 mL polypropylene sterile tubes, placed on ice in a cooler, and were processed immediately upon arrival four days after collection.Different types of media were used to facilitate the growth of the various types of bacteria. Hemolytic bacteria were of interest in this study since they produce hemolysis which is associated with virulence  and exotA total of 93 colonies were selected for further species identification tests. These included the β hemolytic colonies, totaling 50 colonies from the blood agar plates: all 12 colonies from the TCBS agar plates, 22 selected representative colonies from LB based on diversity, and nine colonies from R2A plates. Only one colony of the same color, morphology, surface, and opacity was selected from the R2A plates. The PCR DNA template was prepared using QIAquick PCR purification kits following the manufacturer’s protocol .Universal V3 primers were used for both forward and reverse PCR. The V3 primers amplify the 16 s rRNA. These amplify DNA that codes for a ribosomal subunit, a highly conserved region found in all bacteria. They were selected because of their ability to provide sufficient phylogenic information on bacteria ,22 as thTurbidity measurements were taken using a portable turbidity meter model # 2100P ISO  on site. Turbidity has been used as an indicator of water quality that describes the cloudiness of water caused by suspended particles . Elevate5 CFU/100 mL) was approximately one order of magnitude higher compared to that of closed wells (9 × 104 CFU/100 mL) and MSABI rope pump wells (7 × 104 CFU/100 mL) (p = 0.18). The average total Heterotrophic Plate Count (HPC) for the open wells (7 × 10/100 mL) . There wp-value of 0.20, indicating that there is a twenty percent chance that the results happened accidentally, and therefore are not significant  in any 100-mL sample [E. coli was detected in all open wells at concentrations >154 MPN/100 mL. Only four closed wells out of eleven, and two MSABI rope pump wells out of ten, tested positive for E. coli with an average concentration of <22 MPN/100 mL , Rhodococcus corynebacterioides DSM 20151, and Microbacterium spp. Only one potential pathogen, Gordonia polyisoprenivorans [Of the 93 colonies selected from the 10 wells for species identification by PCR sequencing, 12 were potential pathogens, eight of which came from the open wells: lus spp. ,32,33,34nivorans ,36 was fThis study provides a snapshot of the water quality for these water sources. Based on microbial water quality standards and water turbidity , the datThe objective of this study was to compare the water quality of three different well designs, in order to help guide future efforts in providing affordable and sustainable interventions to improve access to clean and safe water in rural communities. Previously, MSABI had started to convert open wells into closed wells. However, that program was discontinued before this study began. MSABI rope pump wells were assumed to provide far better water quality than the closed wells (open wells converted into closed wells). However, these initial findings suggest that there may not be a significant difference, given that water sources are protected from outside contamination. In the context of rural areas with limited resources and high poverty rates, interventions that the community themselves can afford are critical in improving access to safe and clean water, as well as fostering ownership in a community. Drilling deeper wells requires drilling machines that need to be brought into the community, which is expensive. As we enter the new phase of achieving SDG goals to improve access to safe and clean water, efforts should be made to explore how the communities themselves can participate in these interventions, to achieve sustainability and ownership. Solutions should coordinate and leverage existing community efforts and explore technologies that are feasible, so as to build sustainability beyond donor funding.