All vertebrates possess a thymus, whose epithelial microenvironment is essential for T cell development and maturation. Despite the importance of the thymus for cellular immune defense, many questions surrounding its morphogenesis remain unanswered. Here, we demonstrate that, in contrast to the situation in many other epithelial cell types, differentiation of thymic epithelial cells (TECs) proceeds normally in the absence of canonical Wnt signaling and the classical adhesion molecule E-cadherin. By contrast, TEC-intrinsic activation of β-catenin-dependent Wnt signaling blocks the morphogenesis of the thymus, and overexpression of a secreted Wnt ligand by TECs dominantly modifies the morphogenesis not only of the thymus, but also of the parathyroid and thyroid. These observations indicate that Wnt signaling activity in the thymus needs to be precisely controlled to support normal TEC differentiation, and suggest possible mechanisms underlying anatomical variations of the thymus, parathyroid and thyroid in humans. Despite defying standard classifications for epithelial cells, the epithelial nature of TECs is demonstrated by their expression of a range of keratins, and their embryological origin from the pharyngeal endoderm2.The epithelial microenvironment of the thymus exhibits a unique cellular architecture, which strains the standard definitions of epithelial structures, both in anatomy and function. Anatomically, epithelial cells are typically arranged into stereotypical cell layers; functionally, they are usually categorized as having absorptive, secretory or barrier roles. By contrast, thymic epithelial cells (TECs) are arrayed in a complex, reticulated configuration, and seemingly lack adsorptive, secretory or barrier functions. Instead, the unusual TEC architecture is thought to support the functional role of the thymus - the recruitment of lymphoid progenitors, the induction of T cell development and the quality control of the emerging repertoire of somatically diversified antigen receptors for self-tolerance3. Gcm2 begins to be expressed at ≈E10.5 in the epithelial cells located in the anterior-dorsal region of the pouch and specifies a parathyroid fate, while Foxn1 is expressed slightly later at ≈E11.0 in the epithelial cells of the posterior-ventral region of the pouch, and specifies the adoption of a thymic fate. Exactly how these expression domains are established is not entirely clear, however, with respect to the thymic rudiment BMP6, Shh7 and Wnt signaling8 have all been implicated in the induction of Foxn1 expression.The thymic epithelium arises from the third pharyngeal pouch (3PP). In mice, the 3PP forms at around embryonic day 10.5 (E10.5); it consists of a simple endodermal cell layer divided into two domains, each fated to become the epithelial component of either the thymus or the parathyroid. These domains can be identified by the expression of two key transcription factors, Gcm2 and Foxn1en bloc migration to their target destination in the mediastinum, the thymic lobes increase in size to accommodate a rapid expansion in thymocyte numbers, and become patterned into the stereotypical cortical and medullary domains.After patterning of the 3PP into parathyroid/thymus domains, the parathyroid and thymus anlagen separate and adopt organ-specific architectures and functions. The segregation of the parathyroid and thymic anlagen occurs at approximately the time when the nascent thymic lobes are first populated by lymphoid precursors, and the thymic epithelium transitions from a simple cell layer to a reticulated architecture. Simultaneously with the 17 and signaling molecules25 that regulate thymus and parathyroid specification and positioning have been identified, there is little information regarding the molecular underpinnings of many of the steps involved in the transition from 3PP to mature thymus.While each of these events in thymus development have been extensively documented histologically, and several of the transcription factorsCdh1 gene) is a classical cadherin involved in the formation of adherens junctions; its extracellular domain is composed of cadherin repeats that support homotypic interactions, its intracellular domain interacts with catenins in order to mediate connections with the cytoskeleton26. Previous studies demonstrated that E-cadherin is expressed by TECs27, and that blocking homotypic E-cadherin interactions prevents thymic lobe formation in re-aggregate thymus organ culture (RTOC) assays28, suggesting a functional role in thymus organogenesis. Intriguingly, β-catenin (encoded by the Ctnnb1 gene), a critical binding partner of E-cadherin has also been implicated in thymus development29.E-cadherin ; this outcome is in contrast to the situation in the Foxn1-expressing whisker follicle, where the activity of the reporter is high  and keratin-8 (K8) staining  result of either defects in epithelial Wnt signaling pathway activity, or due to altered cell-cell adhesion via classical cadherins. To investigate if activation of canonical Wnt signaling in TECs can perturb their development, we expressed a stabilized form of β-catenin in TECs , while stochastic events may modify the final outcome . Additionally, although cervical thymic tissue is considered an anomaly in mice and humans, it is worth noting that the position and number of thymi lobes shows considerable inter-species variation, and that cervical thymic tissue is a normal feature of various vertebrate species, such as chickens59, sheep60 and wallabies61. We speculate that species-, strain- and individual-specific eccentricities in Wnt signaling pathways might account in part for the relatively broad phenotypic range of both normal and anomalous thymus anatomy.A striking outcome of this study was the finding that activation of Wnt signaling modulates the migration of not only the thymus, but also the parathyroid and thyroid. When Wnt signaling was activated in a TEC-intrinsic manner, organ morphogenic defects were restricted to the thymus and parathyroid, indicating that disruption of short-range cellular interactions within the common anlage of these two organs is sufficient to cause ectopic development. Moreover, paracrine signals emanating from the thymus have the potential to modulate the development of nearby organs, since morphogenic defects were observed not only for the thymus and parathyroid, but also the thyroid when the secreted ligand Wnt4 was overexpressed by TECs. Interestingly, ectopic thymus and thyroid tissue is occasionally observed in humans, and while typically benign, such ectopic tissue can in some instances be detrimental63, and the ectopic thyroid tissue observed in our study bears a striking resemblance to the pyramidal lobe tissue observed in human embryos64. Additionally, lingual thyroid65, is a known congenital anomaly present in up to 10% of the population66. The detection of pyramidal lobe-like and lingual thyroid tissue in Wnt4- and Noggin-transgenic mice might suggest that similar congenital abnormalities in humans, sometimes presenting as combined thyroid dysgenesis or agenesis with ectopic thymus tissue69 arise due to aberrations in Wnt or BMP signaling pathways during development.Another unusual aspect of the Wnt4- and Noggin-overexpression models developed here was the detection of ectopic thyroid tissue either ventral to the trachea, or within the septum of the tongue. The presence of analogous ectopic thyroid tissue has been described in humans; a pyramidal lobe of the thyroid is a common variation of human thyroid anatomy, estimated to occur in approximately 40% of individualsWnt4-transgenic mice demonstrated reduced cellularity. By contrast, when Wnt4 is overexpressed in hematopoietic progenitors transplanted into adult mice – rather than in the embryonic thymic stroma as is the case in our model - an increase in thymic cellularity is observed, associated with an expansion of lymphoid precursors in the bone marrow70. Interestingly, in hematopoietic cells71, Wnt4 exerts its effect in a β-catenin-independent manner. This may also be true for TECs, since β-catenin-deficiency could not rescue the ectopic location of Wnt4-transgenic thymi, a result corroborated by the observation that the BATgal reporter, which is induced by β-catenin-dependent Wnt signaling42 displayed little activity in thymus. Collectively, this data indicates that Wnt signaling in the thymus predominantly occurs via β-catenin-independent pathway(s)72.In addition to being ectopically located, the thymus in 73, demonstrating that normal development requires the appropriate level of Wnt signaling acting on a number of different target cell types. Several other signaling pathways such as BMP4, FGF74, ephrin20 and retinoic acid (RA)23 pathways have all been shown to modify thymus morphogenesis, raising the possibility that interaction(s) with these pathways may underpin the disturbances caused by activated Wnt signaling. Our observation that blocking BMP signaling exacerbated the morphogenic defects caused by Wnt4-overexpression clearly demonstrates that these pathways can interact to modify migration of pharyngeal endoderm-derived organs.How might Wnt signaling modulate the development and migration of the pharyngeal apparatus? Perturbations in Wnt signaling in mesenchymal cells can result in ectopic thymus and parathyroid developmentIn conclusion, our results indicate that the classical cadherin E-cadherin is not required for the formation of the unique epithelial reticulum of the thymus. It also appears that the extent of β-catenin-dependent and -independent Wnt signaling in TECs must be precisely controlled to ensure normal development. Moreover, the activation of Wnt and inhibition of BMP signaling act as dominant modifiers of organ migration during morphogenesis of the thymus and other pharyngeal derivatives, indicating that these two signaling pathways have antagonistic roles.75, Ctnnb1fl  76, Cdh1fl  77, Foxn1::Cre78, Foxn1::EGFP57, Foxn1::Noggin4 and BATgal42 mice have been previously described. The Foxn1::β-catenin transgene was constructed by cloning a Myc-tagged, stabilised form of β-catenin derived from plasmid pCS2MMBCS33A 6*Myc (a kind gift from Rolf Kemler) under the control of the Foxn1 promoter. The stabilised β-catenin contains four serine-to-alanine phosphorylation site mutations (S33A/S37A/S41A/S45A) and a 6*Myc-tag at the C-terminus. The Myc-tagged, stabilised β-catenin cDNA was cloned into the NotI site of vector pAHB14, downstream of a 27,970 bp Foxn1 promoter fragment (corresponding to nucleotides 5680–33650 of Y12488)4. The Foxn1::Wnt4 transgene was constructed by cloning a Wnt4 cDNA fragment corresponding to nucleotides 43–1101 of NM_009523 into the NotI site of pAHB14. The Wnt4 cDNA was amplified from newborn thymus cDNA with the primers Wnt4_f (5′ GAGGCGGCCGCACCATGAGCCCCCGTTCGTGCCT 3′) and Wnt4_r (5′ GAGGCGGCCGCTCACCGGCACGTGTGCATCTCC 3′), which introduce NotI restriction sites (underlined in the primer sequence). Transgene constructs were injected into FVB/N pronuclei using standard techniques, and the resulting transgenic mice were subsequently backcrossed to a C57BL/6J background.Nlk-deficientFor timed matings, the day of plug detection was designated as E0.5. All animal experiments were performed in accordance with relevant guidelines and regulations, approved by the review committee of the Max Planck Institute of Immunobiology and Epigenetics and the Regierungspräsidium Freiburg, Germany (license Az 35-9185.81/G-12/85).Embryos for H&E staining and ISH were fixed in 4% PFA and subsequently embedded in paraffin using standard techniques. Embryos used for X-Gal staining were fixed in 4% PFA for 1 hr on ice, infiltrated with 16% sucrose overnight, then frozen in OCT for sectioning. All embryo sections were cut in the transverse plane, in all figures dorsal is up, ventral is down. Scale bars are indicated.in situ hybridisation (ISH) on paraffin sections was performed using DIG-labelled probes as described79. Sequences for the various probes used are as follows: Axin2, nucleotides 2077-2877 in Genbank Accession number NM_015732.4; Foxn1, nucleotides 2181-3584 in Genbank Accession number XM_006532266.3; Gcm2, nucleotides 615-1540 in Genbank Accession number NM_008104.2; GzmA, nucleotides 211-1019 in Genbank Accession number M13226.1; Titf1, nucleotides 1471-1788 in Genbank Accession number NM_009385.3. Images were acquired on a Zeiss Axioplan 2 microscope equipped with an AxioCam MRc 5 camera.RNA Sections from paraformaldehyde (PFA)-fixed, paraffin-embedded tissue were de-paraffinised in xylene, re-hydrated, and then subjected to antigen retrieval with Tris-EDTA buffer  in a steamer for 17 minutes. Sections were subsequently washed in PBS-Tween 20 (0.05%) and permeabilised with 1% TritonX-100. Antibody staining was performed at room temperature in staining buffer (PBS supplemented with 4% serum and 0.05% BSA). Sections were stained for 1 hr with primary antibodies (see Table 2 and 0.02% IGEPAL in PBS) for 5 minutes at 4 °C, then washed three times with PBS and covered with X-Gal staining solution  and incubated at 37 °C overnight. Sections were subsequently washed extensively in PBS, and then mounted in Mowiol mounting media for imaging. For whole-mount staining mice were sacrificed, and dissected to ensure adequate penetration of the fixative and staining solutions. After bisection below the diaphragm, the skin covering the cervical region and trunk was removed together with the sternum, and the anterior half of the body was submerged in fixative solution for 30 minutes at 4 °C with agitation, washed through 3 changes of PBS, then submerged in X-Gal staining solution supplemented with 0.01% sodium deoxycholate and incubated at 37 °C overnight with gentle agitation. Stained samples were subsequently rinsed extensively in PBS, then post-fixed with 4% PFA in PBS prior to imaging.Cryosections were fixed with fixative solution , and then stained for intracellular antigens. Antibodies for intracellular staining , neutral protease (200 µg/mL) and DNaseI (500 ng/mL) in RPMI 1640 + 2% FCS for up to 90 minutes at 37 °C with agitation. Following digestion, EDTA was added to a final concentration of 2 mM to disaggregate any remaining cell clumps. Cells were then washed with RPMI 1640 + 2% FCS, and then re-suspended in PBS/BSA for staining. Thymocyte suspensions for CD4/CD8 staining were generated by gently pressing thymic lobes through 40 µm sieves. Cell surface staining (see Table t-tests (two-tailed) were used to determine the significance levels of the differences between the means of two independent samples, considering equal or unequal variances as determined by the F-test. For multiple tests, the Bonferroni correction was applied.Supplementary Information


The aim of the present study was to determine the extent of malnutrition in patients waiting for a liver transplant. The agreement among the methods of nutritional assessment and their diagnostic validity were evaluated.Patients on the waiting list for liver transplantation (n = 110) were studied. The variables were: body mass index, analytical parameters, liver disease etiology, and complications. Liver dysfunction was evaluated using the Child–Pugh Scale. Nutritional state was studied using the Controlling Nutritional Status (CONUT), the Spanish Society of Parenteral and Enteral Nutrition (SENPE) criteria, the Nutritional Risk Index (NRI), the Prognostic Nutritional Index (PNI-O), and the Subjective Global Assessment (SGA). Agreement was determined using the Kappa index. Area under receiver operator characteristic curves (AUCs), the Youden index (J), and likelihood ratios were computed.Malnutrition varied depending on the method of evaluation. The highest value was detected using the CONUT (90.9%) and the lowest using the SGA (50.9%). The pairwise agreement among the methods ranged from K = 0.041 to K = 0.826, with an overall agreement of each criteria with the remaining methods between K = 0.093 and K = 0.364. PNI-O was the method with the highest overall agreement. Taking this level of agreement into account, we chose the PNI-O as a benchmark method of comparison. The highest positive likelihood ratio for the diagnosis of malnutrition was obtained from the Nutritional Risk Index (13.56).Malnutrition prevalence is high and prevalence estimates vary according the method used, with low concordance among methods. PNI-O and NRI are the most consistent methods to identify malnutrition in these patients. •Malnutrition prevalence in patients waiting for a liver transplant is high.•The prevalence of malnutrition varied depending on the method of evaluation.•The overall agreement among the malnutrition assessment methods was low.•PNI-O was the method of assessment malnutrition with the highest overall agreement.•NRI obtained the highest positive likelihood ratio for the diagnosis of malnutrition. The presence of protein-calorie malnutrition is common among patients with liver disease. Malnutrition is one of many possible effects of liver disease.7Great variability in the prevalence of malnutrition has been observed, depending on the method used for assessmentBecause there is not a gold standard, different tools must be used to quantify and classify malnutrition.The European Society for Clinical Nutrition and Metabolism recommends evaluating malnutrition in patients with liver cirrhosis through tests, including subjective global assessment, anthropometry, biomedical impedance, and grip strength evaluation.Following the recommendations and due to the variability among methods, some authors have tried to identify the best tool to assess the nutritional state of these patients. Villalobos et alThis study was performed due to the significance of the nutritional state on morbidity and mortality of patients and the difficulty in determining a nutritional assessment method. We aimed to determine the malnutrition degree in patients on the liver transplant list, study the concordance among assessment methods, and explore the diagnostic validity compared to a benchmark that was chosen because it obtained the best overall concordance.We conducted a cross-sectional study at University Hospital Complex in A Coruña, Spain performed during the period of January 2012 through December 2014.The inclusion criteria were patients over the age of 18 years who were included on the waiting list for liver transplant during the study period and who signed the informed consent.Sample size was limited by both the duration of the study and the number of liver transplants per year. We evaluated 110 patients during the period of this study (2012–2014).This sample size (n = 110) allow us to estimate characteristics related to nutritional state with a precision of ±10% and 95% confidence (α = 0.05), assuming a prevalence of 50% of the variable of interest and 10% losses during follow-up. At the same time, this sample size allows estimation of the pairwise agreement among methods with a precision of ±16.50% and 95% confidence, assuming a Kappa index value around 0.400.The patients were identified with a code, keeping their personal data confidential. To obtain the necessary information for the study, clinical records were reviewed, a physical examination was performed, and the patients were interviewed.1.From clinical records: demographic data (age and gender), analytical parameters , transplant etiology  and the presence of hepatic complications 2.From physical examination: patient weight, height, and BMI3.From interviews: the SGAThe following variables were obtained by the time of inclusion on the waiting list for liver transplantation:The degree of liver dysfunction was evaluated using the Child–Pugh Scale,The CONUT and PNI-O scores were selected because they have been shown to be useful tools to assess the nutritional state in patients with chronic liver disease.Finally, the SGAAlthough some of these methods could be considered as nutritional screening tools , while others could be considered as assessment tools (SGA), all of them allow us to calculate the risk of malnutrition.Descriptive analysis was performed for all variables. Continuous variables were reported using means and standard deviations (SD) or medians and interquartile ranges, as appropriate. For dichotomous/categorical variables, absolute numbers and percentages were computed, together with their 95% confidence intervals.Concordance between the different scales that assess nutritional state was studied with the statistical Kappa (K) index. The overall Kappa was estimated, and the homogeneity test was performed to study the agreement amongst methods.PNI-O was the method of the highest level of overall agreement. Taking this level of agreement into account, we chose the PNI-O as a benchmark method of comparison.Diagnostic validity was assessed using the area under the curves (AUCs), the Youden index, sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios. The results of the CONUT and the NRI were compared with PNI-O diagnosis of malnutrition as the reference criteria and displayed on a receiver operating characteristic (ROC) curve. For each method, the ROC AUC was computed, and its optimum cut-off point was determined using the Youden index. The Youden index (J) is defined as the maximum vertical distance between the ROC curve and the diagonal or change line and calculated as J = max (sensitivity + specificity − 1).The study complies with the principles laid down in the Declaration of Helsinki. Informed consent was obtained from all the participants in the study. Confidentiality was preserved in accordance with the current Spanish Data Protection Law (15/1999). This project was approved by the corresponding ethics review board .2 in 58.2% of them. Mean Child–Pugh score was 8.37 , and 48.6% of the patients showed a significant functional commitment (Child–Pugh B). The main etiology was alcoholic cirrhosis (52.7%). The most frequent liver disease complication was ascites (79.1%)  years, and most were men (72.73%). The BMI was ≥25 kg/m (79.1%) .Table 2BThe percentage of malnutrition at the time of inclusion in the transplant waiting list  changed Due to the variability in the prevalence of malnutrition depending on the method used, we calculated the level of agreement among different methods. Pairwise agreement among the methods ranged from K = 0.041 to K = 0.826, with an overall agreement of each criteria with the remaining methods between K = 0.093 and K = 0.364 . PNI-O wThe Youden index, corresponding cut-off points, sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios are shown in Using the NRI and the CONUT (quantitative methods), the cutoff point for malnutrition in patients with liver disease was found to be higher compared to the general cutoff point. The ROC curves for the methods of malnutrition analysis are displayed in From the ROC curves, the optimal cut-off points of these tests for diagnosing malnutrition in the total sample were 98.39 for the NRI and 3.50 for the CONUT, with sensitivities of 96.9% and 75.0%, respectively. The positive predictive values for the NRI and the CONUT were 98.9% and 96.0%, respectively. Negative predictive values were 81.3% and 31.4%, respectively. The NRI was found to have the highest positive likelihood ratio (13.56).It is important to know the nutritional state of the patient, as it is a risk factor for morbidity and correlates with the severity of liver dysfunction,17Among the different evaluation methods, anthropometric parameters, such as BMI, are used. In this study, there were no patients with malnutrition according to BMI, and more than half of the subjects were classified as overweight or obese. This result may be due to most of the patients having ascites, which involves a change in body composition. This finding is similar to those of other publications, which conclude that BMI underestimates malnutrition and is not a suitable method to evaluate these patients.17The methods used in this study can be distributed into three groups: a) structured tests ; b) methods using only analytical parameters (CONUT and PNI-O); and c) methods using analytical and anthropometric methods (SENPE criteria and the NRI). The SGA and SENPE criteria showed lower prevalence estimates of malnourished patients . The low prevalence of malnutrition estimated using the SGA is similar to that reported in other publications,We found a similar prevalence of malnutrition using the SENPE criteria to that reported by Villalobos et al.The NRI, CONUT, and the PNI-O all showed higher malnutrition prevalence  than SENPE criteria and SGA. This can be explained by the pathology of patients, which affects the values of albumin, cholesterol (which is synthesized by the liver), and lymphocytes . In addition, in this study, patients with mild malnutrition were considered malnourished, while only moderate and severe malnutrition patients were considered malnourished in other publications.This study shows that the concordance among the methods of nutritional assessment is not very high . The con29The concordance between the CONUT and the NRI was moderate and similar to the findings of Gimeno et al (K = 0.547). But the results of SENPE criteria with the NRI and the CONUT were weak for both of them and lower than the results obtained by Gimeno et al, with K values of 0.609 and 0.593, respectively.Our results show a lower concordance than that reported in other publications. These findings may be related to the inclusion criteria, because we included patients with liver cirrhosis who were not considered in other published articles.29In methods that also include anthropometric parameters (SENPE and NRI), a lower prevalence of malnutrition was observed in comparison with the CONUT and PNI-O because the increase of weight is considered as an indicator of improved nutritional state. This situation is not realistic in our sample because most of the patients had hydropic decompensation (ascites and/or edema).The method that showed the lowest level of agreement was SGA. This is reasonable, since it is the only method of those considered that does not include anthropometric and/or laboratory parameters. Furthermore, other authors have also demonstrated that SGA is not sufficient as a nutritional screening tool for patients with liver diseases,The overall Kappa values, from highest to lowest, were for the PNI-O, the SENPE criteria, the NRI, and the CONUT. We decided to use the PNI-O as benchmark to determine the diagnostic validity of the other assessment methods because it showed the highest concordance.Although this study was not designed to identify a gold standard for malnutrition assessment in liver disease patients, we explored the results obtained to determine the diagnostic validity of each of the methods considered with respect to the method that showed the highest consistency (the PNI-O). Although the existence of reference bias cannot be ruled out, the results could be useful to identify suitable methods of assessing malnutrition in these patients.Using the PNI-O as the reference method, the methods that showed a higher diagnostic validity for malnutrition according to the Youden Index were the NRI, SENPE criteria, and CONUT. The methods that can be considered good tools to identify malnutrition, due to having high sensitivity, are the SENPE criteria and the NRI . The methods that best identify the nourished patients, due to having high specificity, are the NRI and the CONUT .Likelihood ratios were calculated to assess the accuracy and validity of the diagnostic test and to aid in selecting an appropriate diagnostic test. Moreover, this statistic has advantages over sensitivity and specificity because it is less likely to change with the prevalence of diagnosis.The highest positive likelihood ratio (LR) for the diagnosis of malnutrition was obtained from the NRI (13.56). According with the level of evidence of Canadian Evidence-Based Medicine, when a test has a positive LR > 10, it is considered a good test to confirm the diagnosis of malnutrition.These findings demonstrate that the NRI is highly accurate in the diagnosis of undernutrition in our sample, being the method with the best agreement with the PNI-O, with a value Kappa of 0.846.The present study found high prevalence of malnutrition among patients on the waitlist for a liver transplant and variability in the estimated prevalence of malnutrition depending on the method of evaluation. Moreover, the nutritional assessment methods do not show good concordance. The most valid methods for identifying malnutrition in patients with liver disease are the PNI-O and the NRI.None declared.MTGR, MCPV, BLC and SPF participated in the design and coordination of the study. BLC, SPD and MTSP are the biostatistician of the study. MTGR, MCPV, BLC, AOF, FSL, MGG and SPF reviewed the study protocol and made suggestions that improved the design. All of these individuals are involved in the management of the study. MTGR and MCPV obtained the data for the study.MTGR, MCPV, BLC and SPF drafted the manuscript. All the authors read, revised, and approved the final manuscript.


Plasmodium malariae that infects humans remains indistinguishable from Plasmodium brasilianum, which is widespread among New World primates. Distributed throughout tropical Central and South America, the Callitrichidae are small arboreal primates in which detection of natural Plasmodium infection has been extremely rare. Most prior screening efforts have been limited to small samples, the use of low-probability detection methods, or both. Rarely have screening efforts implemented a longitudinal sampling design. Through an annual mark-recapture program of two sympatric callitrichids, the emperor (Saguinus imperator) and saddleback (Saguinus fuscicollis) tamarins, whole blood samples were screened for Plasmodium by microscopy and nested PCR of the cytochrome b gene across four consecutive years (2012–2015). Following the first field season, approximately 50% of the samples collected each subsequent year were from recaptured individuals. In particular, out of 245 samples from 129 individuals, 11 samples from 6 individuals were positive for Plasmodium, and all but one of these infections was found in S. imperator. Importantly, the cytochrome b sequences were 100% identical to former isolates of P. malariae from humans and P. brasilianum from Saimiri sp. Chronic infections were detected as evidenced by repeated infections (7) from two individuals across the 4-year study period. Furthermore, 4 of the 5 infected emperor tamarins were part of a single group spanning the entire study period. Overall, the low prevalence reported here is consistent with previous findings. This study identifies two new natural hosts for P. brasilianum and provides evidence in support of chronic infections in wildlife populations. Given that callitrichids are often found in mixed-species associations with other primates and can be resilient to human-disturbed environments, they could contribute to the maintenance of P. malariae populations if future work provides entomological and epidemiological evidence indicating human zoonotic infections.There is an increased interest in potential zoonotic malarias. To date,  Plasmodium) were first discovered as early as 1880 [Plasmodium species infecting humans are also circulating in nearby simian and ape communities. Whether such non-human primate host can act as a reservoir of human malarias is a matter of great interest.In 2015 malaria was diagnosed in approximately 212 million people, and resulted in the loss of 438,000 lives worldwide [Plasmodium have been documented in nonhuman primates [Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, and Plasmodium malariae) frequently occur in humans. Along with Plasmodium ovale, Plasmodium species that infect humans do not form a monophyletic group [P. falciparum and P. vivax) are part of larger clades of species that include many that parasitize nonhuman primates [According to the Global Mammal Parasite Database, 27 species of primates , three oic group . The twoprimates ,7,8.Plasmodium brasilianum was first described in monkeys in the beginning of the 20th century and has now been documented in approximately 31 species of New World monkeys [P. brasilianum and Plasmodium malariae that infect humans [P. malariae. Of these, 12 sequences were 100% identical to P. brasilianum strains recovered from howler monkeys in French Guiana, and the remainder were 99–100% identical to P. malariae strains from Myanmar and Papua New Guinea. Although the 18S gene does not allow us to accurately discern recent host switches due to its rate and semi-concerted mode of evolution [Plasmodium. The only factor that has ever been used to differentiate these two parasites is host-identity (human or not), and yet experimental studies have demonstrated that nonhuman primates are susceptible to P. malariae [P. brasilianum and P. malariae could very well be the same organism.In South America,  monkeys ,10. Thist humans ,11–14. Lt humans  collectevolution , the genmalariae . These fP. malariae/brasilianum causes quartan malaria, so termed for having 72-hour erythrocytic cycles, unlike P. falciparum or P. vivax that cycle every 48 hours [P. malariae has led to nephrotic syndromes in humans and experimentally infected monkeys [P. malariae/brasilianum can occur when hosts are subjected to stressful conditions or become immunocompromised [P. malariae/brasilianum with other Plasmodium spp. are common [P. malariae/brasilianum is relatively low, co-infections are probably under-detected when screenings are performed only by microscopy [48 hours . P. mala monkeys ,18, and  monkeys ,20. Recrpromised ,21. In me common ,22,23. Scroscopy ,18, and Plasmodium qualifies P. malariae as an important health concern at the human-wildlife interface given that present research suggests it is the same as P. brasilianum. Fundamental to assessing health risks are the development of a clear understanding of host breadth and associated prevalence for these (or this) species. The majority of infections found in wild New World monkeys are confined to the Atelidae, Pitheciidae, and Cebidae, while detection in the Callitrichidae, perhaps the most speciose and widespread Neotropical primate family, remains rare [Plasmodium infections, these characteristics of the callitrichids might implicate them as an important sylvatic component for malaria control efforts.The ability to reside in non-human primates, induce renal pathology, persist as a chronic infection in humans, and interact with other species of ins rare ,10,14,22ins rare ,25. Unliins rare –28. Moreins rare . If provPlasmodium infections from wild populations of callitrichids to date, several biases stand out. First, although five of the seven callitrichid genera have been tested for P. brasilianum , the majority of species have not been sampled [Saguinus midas, Saguinus niger, Saguinus geoffroyi, Saguinus martinsi, Mico humeralifer, Leontopithicus chrysomelas, Leontopithecus chrysopygus, Leontopithecus. rosalia, and Callithrix geoffroyi. However, only the first three of these species  are clear cases of infection in natural environments; the other infections were recently detected from captive animals at primate research or rescue centers [S. geoffroyi [S. niger [S. niger [S. midas [P. brasilianum tends to exhibit low prevalence, averaging 0.045±0.043 for the Callitrichidae and 0.023±0.024 for the Primate Order[P. malariae/brasilianum and may act as a reservoir for human malaria.When considering accessible survey data on  sampled ,10. Of t centers . Of the eoffroyi , 4/109 fS. niger , 4/178 iS. niger ,33, and S. midas . This isPlasmodium infections longitudinally across four years in two sympatric species of callitrichids, the saddleback tamarin, Saguinus fuscicollis weddelli , andpecifics . Second,Saguinus fuscicollis) and emperor (Saguinus imperator) tamarins at the Estación Biológica Rio Los Amigos (EBLA) in the Madre de Dios Department of southeastern Perú  (terra firme and várzea habitat.Samples were collected from a free-ranging population of saddleback (05’57”W) . This prSince 2009, we have encountered approximately 70 unique individuals each year, across both species. Our program is optimized to ensure habituation of primates to subsequent human observation . AnimalsImmediately after blood draw, blood smears were made on standard microscope slides and air-dried. All smears were fixed for five minutes in 100% methanol within six hours and stained in Giemsa’s solution following Valkiunas et al.  within t20 using standardized protocols recommended by the manufacturer . DNA samples from the first three years (2012–2014) were screened for haemosporidian parasites by a nested polymerase chain reaction (nPCR) protocol that targets part of the parasite cytochrome b (cytb) gene, 709 base pairs (bp), following Duval et al. [cytb gene  for infected individuals across the entire study period (2012–2015), we employed a separate nPCR protocol that amplifies a 1,038 bp fragment with specific forward-TGTAATGCCTAGACGTATTCC and reverse-GTCAAWCAAACATGAATATAGAC primers for the outer PCR and forward-TCTATTAATTTAGYWAAAGCAC and reverse-GCTTGGGAGCTGTAATCATAAT primers for the inner PCR, following Pacheco et al. [2, 1X PCR buffer, 1.25 mM of each deoxynucleoside triphosphate, 0.4 mM of each primer, and 0.03 U/μl AmpliTaq polymerase . The PCR conditions were: a partial denaturation at 94°C for 4 min, 36 cycles of 1 min at 94°C, 1 min at 53°C and 2 min extension at 72°C, and a final extension of 10 min added to the last cycle. Then, a nested PCR using 1 μl of the first amplification as the template was performed under identical PCR conditions. After electrophoresis, all amplified products were excised from the gels, purified by the QIAmp Gel Extraction Kit (Qiagen), and both strands were sequenced using an Applied Biosystems 3730 capillary sequencer.DNA was isolated from a 3 mm diameter hole punch from the blood stored on Whatman FTA Micro Elute Cards into 30ul of ddHl et al. . To confo et al. . PCR ampcytb gene sequence identity for samples positive for Plasmodium was confirmed using BLAST against NCBI. Electropherograms were visually examined to rule out mixed infections. In addition to the sequences obtained in this study, we included a total of 26 sequences available in GenBank for Plasmodium parasites isolated from mammals in the subsequent phylogenetic analysis. The phylogenetic relationships between sequences were inferred on the cytb gene using MrBayes v3.2.6 with the default priors [6 Markov Chain Monte Carlo (MCMC) steps, and after convergence was reached , we discarded 25% of the samples as burn-in [Complete t priors . Alignmet priors  with mant priors . Bayesia burn-in . Then, t burn-in .Saguinus fuscicollis, 92 from Saguinus imperator) spread across 129 individuals  during the study period. Zero infections were confirmed from examination of thin blood smears; however, 10 samples were successfully amplified by nPCR from emperor tamarins  and one from a saddleback tamarin in 2014  were 100% identical to each other and to reference sequences for human isolates of P. malariae and squirrel monkey (Saimiri sp.) isolates of P. brasilianum , 0.13 in 2013 (n = 24), and 0.10 in 2014 (n = 21), and 0.08 in 2015 (n = 26) with an average across years of 0.11+/-0.03 . While pPlasmodium species other than P. malariae/brasilianum can infect both human and nonhuman primates. However, in many cases, there is still limited evidence that non-human primates are a reservoir of human malaria. A clear example of zoonotic malaria is Plasmodium knowlesi, a simian parasite in southeast Asia that has been repeatedly found in humans [Plasmodium cynomologi parasitizes Asian macaques and has at least one documented case in humans [P. vivax is suspected of circulating in a subset of west African apes that are positive for the Duffy blood group antigen molecule [Plasmodium simium are large New World monkeys (Atelidae), but there is at least one case of a human infection [P. falciparum, the most virulent human Plasmodium, is sometimes detected in New World monkeys (8 species over 5 genera) [A handful of n humans ,49. Thisn humans . In addin humans . Beyond nfection . P. falcP. falciparum has been found in apes, particularly chimpanzees [P. knowlesi from isolated macaque (Macaca spp.) populations in Borneo and Peninsular Malaysia [P. malariae/brasilianum, however, is to better characterize its host range throughout South America.Although host switches are common in non-human primates, not all host switches indicate the presence of a zoonosis ,54. As ampanzees ,55. Howempanzees . Thus, iMalaysia . A firstP. brasilianum has been screened for in S. fuscicollis on two separate occasions between 1995 and 2013  in Brazil using only microscopy [S. imperator (n = 2) [P. brasilianum/malariae. Like past studies of Plasmodium from other simian hosts in South and Central America, P. brasilianum was genetically identical to P. malariae using cytb, reinforcing that they are likely to be a single organism [croscopy , and onl (n = 2) , with zeorganism . That weP. brasilianum occur in the wild, consistent with the low, stable prevalence in emperor tamarins despite decreasing incidence of new infection  and to new groups. This also opens possibilities for measuring individual health parameters before and after the onset of P. brasilianum infections and whether there exist associations with other natural parasites [Importantly, our data suggest that chronic infections of nfection . If truearasites .Plasmodium from Neotropical primates make little mention of how parasites are distributed within host populations, but potentially uneven distributions would be an important factor for assessing disease risk [Plasmodium inui, also a quartan parasite, does not show host population structure in Borneo [P. knowlesi does in Malaysia [Plasmodium infection. Emperor tamarins generally sleep in thick tangles of branches and vines, and sometimes tree holes, and although this is an unlikely explanation, it will be worth ruling out in future years. Second, there could be additional hosts within the home range of the infected group of S. imperator that might increase the parasite encounter rate. The latter scenario is not unlikely, as there are 9 other nonhuman primate species present that could host Plasmodium, including members of the Atelidae, Cebidae, Pithecidae, and Aotidae, as well as a small but dynamic population of human researchers. However, all of the nonhuman primate species occur concurrently throughout the study area, and thus may not explain clustering within one species and one social group. Regarding the risk from infected humans at EBLA, the home range of this group does not overlap with the stations basecamp. Of two saddleback and two emperor tamarin groups with home ranges that do intersect with the basecamp, only the sole saddleback tamarin that appeared infected for a single year is a member of these groups. That these groups closest to basecamp, which are the most well-sampled and exposed to the highest degree of proximity with researchers, accounted for a single Plasmodium infection from one year, suggests that transmission from human to nonhuman primates is not the source of P. brasilianum infection at EBLA. However, none of the resident staff or researchers were tested for infection even though asymptomatic human carriers of P. malariae have been reported [P. malariae in human populations that are in contact with non-human primates infected with P. brasilianum are needed to fully assess whether there are zoonotic infections. Finally, there could be differences in host susceptibility or simply very low parasitemia below the detection of the PCR implemented in this study. As we have only sampled tamarin groups that occur within an approximate 200-hectare area, it would be worthwhile to expand the study area to see if other clusters are present.The parasite prevalence we observed for emperor tamarins was in the same range that has been published from other wild Neotropical primate populations. Although prevalence was too low to analyze variation between different demographic groups, we observed that 4 of 5 infections occurred in a single group (out of 7). Previous studies on ase risk , particun Borneo  but P. kMalaysia . This efMalaysia  found prreported ,60, and


To assess the influence of the Yellow River, 31 sites are selected from the wheat-maize double cropping system, and grouped into two: 10 sites near and 21 sites far from the river. Our data show that mean soil C:N ratio is low (7.6–9.9) across the region, and not affected by the Yellow River. However, SOC and TN are significantly (P < 0.05) lower in subsoil near the Yellow River (2.0 and 0.2–0.3 g kg−1 for SOC and TN) than far away (3.1 and 0.4 g kg−1); δ13C is significantly more negative below 60 cm near the river (−23.3 to −22.6‰) than far away (−21.8 to −21.4‰). We estimate that the contributions of wheat and maize to SOC are 61.3–68.1% and 31.9–38.8%, respectively. Our analyses indicate that the overall low levels of SOC in the North China Plain may be associated with the low soil C:N ratio and less clay content. The hydrological processes may also partly be responsible, particularly for those near the Yellow River.To better understand the characteristics of soil organic matter (SOM) in the North China Plain, we evaluate the large scale variations of soil organic carbon (SOC), total nitrogen (TN), carbon to nitrogen (C:N) ratio and stable carbon isotopic compositions (δ High SOM content often indicates high soil fertility and thus high crop production4. On the other hand, soil organic carbon (SOC) is a large reservoir for carbon, playing an important role in the global carbon cycle and climate mitigation5.Land is an essential nature resource for sustainable agriculture. Maintaining good soil quality is important for high grain production. Soil organic matter (SOM) is a key index of soil fertility, which is closely correlated with crop yields6. The North China Plain and northwest China occupy 44% of the nation’s maize production area and produce 50% of maize grain7. Given the fact of relatively larger land with lower soil fertility (thus lower rates of production) in the northwest China, maize yield in the North China Plain would be much above average. Based on these analyses, one may expect that soil fertility thus SOM or SOC in the North China Plain should be above average of China.The North China Plain has a long history of intensive cropping, and is an important grain production base in China. This region counts 57% of the nation’s wheat-grow land and provides 66% of the nation’s wheat productivity−1) was lower than the average (11.3 g C kg−1)8. Our recent studies revealed that mean SOC content was lower than 9.5 g C kg−1 in the upper 20 cm of the Hebei Plain and the upper Yellow River Delta9. Some other studies carried out in the North China Plain also showed relatively low SOC contents (<9 g C kg−1)12.However, the data from the Second National Soil Survey (conducted in the 1980s) indicated that the SOC content in topsoil of the Northeastern China , which can affect soil condition, nutrients cycling and crop growth. For instance, soil C:N ratio has influences on microbial activities thus on SOM decomposition rate19, soil health and biogeochemical cycles13.Apart from SOC, total nitrogen (TN) is another important index that can indicate soil nutrient condition, with influence on the nitrogen cycle and crop growth13C abundance (δ13C) in SOC is another characteristic of organic matter, which is largely related to sources of organic carbon, and also affected by decomposition21. Therefore, the isotopic technique has been widely used to study the history of land use and trace the sources of SOC by quantifying relative contributions of C3 and C4 plants that have different isotopic 13C signatures25. Limited studies have employed this approach to quantify the contributions of wheat and maize to SOC in the northern China27.The natural 28. There were evidence that high soil salinity inhibits organic matter decomposition30, thus one may assume that the SOM level would be greater near the Yellow River. On the other hand, the hydrological cycle that is primarily influenced by the river water and underground water may also have implications for the biogeochemical cycle 31. Studies have showed that the Yellow River transports various forms of carbon including dissolved organic carbon (DOC), other chemical materials and biological materials from the land to the Bohai Sea32. Underground water table is shallow near the Yellow River, implying stronger hydrological impacts near the river33.A significant part  of the North China Plain is largely influenced by the Yellow River through the hydrological cycle. Our recent study showed that soils near the Yellow River contain higher levels of salts relative to those far away from the river13C) in the upper 100 cm of the North China Plain. To evaluate the influence of the Yellow River, we collect soil samples from the same cropping system , and separate them into two groups: 10 sites near and 21 sites far from the river. The objectives of this study are to better understand the characteristics of SOM in the typical wheat-maize rotation system, and to investigate the impacts of the hydrological processes in association with the Yellow River on soil fertility of the North China Plain.In this study, we analyze large scale dynamics of SOM properties  and water-soluble Mg2+ (26 to 65 mg kg−1). However, mean soil EC and TDS present an increasing trend over depth, i.e, from 271 μs cm−1 in 0–20 cm to 445 μs cm−1 in 80–100 cm for EC, and from 681 to 1117 mg kg−1 for TDS. For the area far away from the Yellow River, our data reveal a small range for pH (8.5–8.7), water-soluble Ca2+ (51–72 mg kg−1) and Mg2+ (16–22 mg kg−1) over the 0–100 cm; average EC increases significantly from 141 (above 20 cm) to 299 μs cm−1 (below 80 cm), and TDS from 354 to 846 mg kg−1  is larger near the Taihang Mountain and in the upstream of the Yellow River in the study area Fig. . There i−1) than far away (3.1 g kg−1). Mean TN below 20 cm is lower close to the river (0.24–0.46 g kg−1) than far away (0.42–0.48 g kg−1), and the difference is significant below 60 cm   to −22.5‰ (80–100 cm), while δ13C far away from the river shows little vertical difference (ranging from −21.4 to −21.8‰).The δer) Fig. . Similarway Fig. . A signi−1) in the topsoil is relatively low, but TN value (0.7–1.6 g kg−1) is not so low34. For example, SOC level in the alluvial loess of the North China Plain is lower than in the deposited loess of the Loess Plateau (11.3–11.5 g kg−1), but TN is higher relative to those (0.9–1.0 g kg−1) in the Loess Plateau34. Similarly, SOC is lower in the North China Plain relative to Northeast and South China (8.9–25.4 g kg−1) with high yields, but TN is comparable (1.0–1.2 g kg−1 for Northeast and South China)17. The decoupling between SOC and TN will not only lead to variations in soil C:N ratio in the cropland, but also has implications for nitrogen release in association with decomposition of SOM.Given that both SOC and TN are important parameters for croplands, we compare both with other studies Table . Based o35. However, soil C:N ratio is considerably lower in our study than those in the black soil with maize cropping (12.3) in Songnen Plain (Northeast China) and red soil with wheat-maize cropping (11.3) in Qiyang (South China)17. There is evidence that SOM decomposition rate has a negative relationship with soil C:N ratio36. Thus, the low soil C:N ratio in the North China Plain may be partly responsible for the low levels of SOM.Mean soil C:N ratio over 0–20 cm in our study is 9.4, which is within the range of 6.4–10.0 reported for the cropland of the North China Plain with the same soil type and cropping system  reported for other wheat-maize croplands of North China38. Overall, δ13C shows a little vertical change in the area far from the Yellow River (−21.4‰ to −21.8‰), but becomes more negative with depth near the river. However, limited studies reveal an enriching trend over depth, i.e., in the wheat-maize croplands of Quzhou, Zhengzhou and Yangling38.The studies on δC3) and maize (ƒC4) to SOC in the North China Plain, using the two end-member mixing model39:13CSOC represents the stable 13C composition in SOC, δ13CC3 in wheat and δ13CC4 in maize. According to Boutton40, average δ13C in wheat and maize are approximately −27‰ and −13‰, respectively.To better understand SOC dynamics in the wheat-maize rotation system, we quantifies the relative contributions of wheat  reported that the contribution of wheat to SOC is 60–72% in other wheat-maize double cropping systems of North China27. A long-term experiment in wheat-maize cropland of Mexico (with similar climate) also indicated greater contribution to SOC by wheat residues22. Earlier studies suggest that maize residue has a rapid decomposition rate, probably resulting from the low lignin content and low C:N ratio and relatively higher temperature during the growing season41.Wang 13C in subsoil. In particular, mean content of SOC below 80 cm and TN below 60 cm are significantly lower near the river than far away .We first assess the possibility of enhancement in SOM decomposition. The clay fraction is relatively higher near the river, which makes decomposition of SOM difficult due to protection by aggregationer Table , which w43, implying stronger hydrological processes near the river. Apparently, the hydrological influence on the subsoils would be more intensive than the topsoils, due to the linkage to the underground water. Based on the above analyses, we postulate that the hydrological processes  may be partly responsible for the lower SOC and TN contents observed in the subsoils near the river. While there is no direct measurement of relevant variables, there is evidence of significant amounts of organic carbon in the Yellow River32, which could indirectly support our inference.We then evaluate the possibility of the hydrological impacts. There is evidence that precipitation near the river (651 to 750 mm) is more abundant than far from the river (551 to 650 mm)44, and the river runoff is profound in summer45. The associated hydrological processes during the summer maize’s growing season would lead to more maize residues removed thus relatively more wheat residues remaining in soil profiles. The more negative δ13C in the subsoil near the river confirms that there is more wheat derived SOC. Such hydrological impacts would exist widely in the North China Plain, but be more pronounced near the Yellow River, which is supported by our data and an earlier study27 that demonstrate more negative δ13C values in the soil profiles near the Yellow River. More studies that involve hydrological processes are needed to better understanding the underlying mechanism regulating the carbon cycle in the cropland of North China Plain.Precipitation occurs mainly during the period of July-August in the North China Plain13C in SOC over 0–100 cm in the representative wheat-maize cropland of the North China Plain. Compared with other croplands in China, SOC is relatively lower, but TN comparable in the North China Plain. As a result, soil C:N ratio is relatively small (9.4) in the North China Plain. The contribution of wheat to SOC (61.3–68.1%) is almost double of the maize’s contribution (31.9–38.8%).We have investigated spatial large-scale distributions of SOC, TN, soil C:N ratio and δ13C value is more negative near the river than far away. Our analyses imply that the low levels of SOC in the North China Plain may be partially owing to the relatively low soil C:N ratio, and the low SOC in the subsoils near the Yellow River could be linked to the hydrological processes .There are significant differences in the subsoils’ characteristics between the near river and far away groups. SOC and TN contents are lower, and δ43. The elevation in the study area shows little variation, ranging from 63 m to 4 m. The studying area covers almost 80 thousand square kilometer, but the soils are all developed on alluvial loess. The soils are Ochri-Aquic Camosols and Endorusti—Ustic Cambosols according to the Chinese soil classification system (1995), which are classified as the Calcaric Cambisol and Fluvo-aquic in the FAO-UNESCO system (1988)9. The groundwater table is gradually increasing, and salinity in the shallow groundwater and soil is increasing from the west plain to east coastal plain33. The main cropping system is a double rotation of winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.). Based on our interviews with farmers, agricultural management  are very similar in the study area. Farmers often use mixed mineral nitrogen-phosphorus-potassium fertilizers, and irrigate with underground water in the area far away from the Yellow River and with groundwater and river water near the Yellow River.The studying area, the main part of the North China Plain, is characterized by a typical continental temperate monsoon climate, with a mean annual air temperature ranging from 12 to 14 °C and precipitation from 550 to 750 mm. The rainfall, largely concentrated in the season of June-September, increased from northwest to southeast28 and 24 sites in Hebei Plain in early October 20159. In this study, we chose representative sites: 10 sites near the Yellow River and 21 sites far away. At each site, 3-4 soil profiles were randomly selected. We collected soils over 0–20, 20–40, 40–60, 60–80, and 80–100 cm (using 5-cm diameter soil augers). Soil samples were air–dried, well mixed and sieved to pass a 2-mm screen. The determinations of pH, EC, TDS, soluble Ca2+ and Mg2+, SOC and TN were reported by Shi et al. (2017)9. Stable 13C isotope was determined by measuring the isotopic composition of collected CO2 using a Finnigan MAT Delta Plus XP Isotope Ratio Mass Spectrometer. The analyses of total soil nitrogen, SOC and stable δ13C isotope were performed at the State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences. We reported isotope data in delta notation relative to the Vienna Pee Dee Belemnite (VPDB).In our previous works, we sampled at 31 sites in the upper Yellow River Delta in August 201513C, wheat-SOC and maize-SOC. All analyses and figures were performed using SPSS (version 22), Sigmaplot (version 12.5) and Arcgis (version 10.1).We used Fisher’s protected least significant difference (LSD) to compare all parameters between different soil layers, including SOC, TN, soil C:N ratio, δ


Despite concerns about the degree of compassion in contemporary healthcare, there is a dearth of evidence for health service managers about how to promote compassionate healthcare. This paper reports on the implementation of the Creating Learning Environments for Compassionate Care (CLECC) intervention by four hospital ward nursing teams. CLECC is a workplace educational intervention focused on developing sustainable leadership and work-team practices designed to support team relational capacity and compassionate care delivery.To identify and explain the extent to which CLECC was implemented into existing work practices by nursing staff, and to inform conclusions about how such interventions can be optimised to support compassionate care in acute settings.Process evaluation guided by normalisation process theory. Data gathered included staff interviews (n=47), observations (n=7 over 26 hours) and ward manager questionnaires on staffing (n=4).Frontline staff were keen to participate in CLECC, were able to implement many of the planned activities and valued the benefits to their well-being and to patient care. Nonetheless, factors outside of the direct influence of the ward teams mediated the impact and sustainability of the intervention. These factors included an organisational culture focused on tasks and targets that constrained opportunities for staff mutual support and learning.Relational work in caregiving organisations depends on individual caregiver agency and on whether or not this work is adequately supported by resources, norms and relationships located in the wider system. High cognitive participation in compassionate nursing care interventions such as CLECC by senior nurse managers is likely to result in improved impact and sustainability. Despite renewed focus on compassion in UK healthcare and internationally, there is a lack of agreement on how best to promote and sustain compassionate healthcare.Recent years have seen the development and evaluation of a number of interventions focused on improving compassionate care at team rather than individual practitioner level.Creating Learning Environments for Compassionate Care (CLECC) is a team-based workplace educational programme focused on creating a sustainable ‘expansive’ learning environment, through leadership and team practices , that enhances team capacity to provide compassionate care .19–22 ThCLECC’s 4-month implementation period, facilitated by a senior practice development nurse (PDN), includes a combination of set activities that represent expansive workplace practices, with a view to embedding these practices to support sustainability : regularexploring how and in what ways the new practice was initially received, how individually and collectively people practically conceptualise and make sense of it (coherence)assessing the degree of ownership of and participation in the new practice by key individuals and teams (cognitive participation)identifying the work that individuals and teams do to enact the new practice (collective action)exploring the perceived impact of the new practice on staff work and on patient outcomes (reflexive monitoring).This qualitative process evaluation used NPT to identify and explain the extent to which CLECC was implemented into existing work practices and to identify how CLECC can be optimised to support sustained compassionate care delivery in acute settings. NPT focuses on four dynamic processes  that motivate and shape implementation processes. Part of a wider feasibility study of CLECC,The CLECC intervention was introduced to four inpatient wards in two general hospitals in England in 2015. Wards with high proportions of older patients in which the ward manager was expected to remain in post for at least 6 months were recruited through ward manager agreement. Wards specialised in either medicine for older people (n=3) or trauma and orthopaedics (n=1). Data were gathered between May 2015 and May 2016.Individual face-to-face semistructured interviews were undertaken with staff over a 12-month period beginning at the outset of the implementation period followed by two further interview rounds (at 3–6 months and 7–12 months). We purposively sampled volunteers to capture variations in staff grade and ward. Recruits were invited to further interviews so that variations over time could be tracked. Where there was attrition, new individuals were recruited to ensure ward and grade variation was maintained. Senior nursing managers were invited to one interview in the final phase. Interview schedules reflected NPT dynamic processes and changed over time to reflect implementation stage. Interviews were conducted by university researchers and lasted on average 46 min. Interviews were audiorecorded, transcribed verbatim and transcripts checked for accuracy. Each PDN, seconded to the role from existing NHS employment, kept detailed field notes. A sample of CLECC learning activities was also observed by university researchers (n=7 over 26 hours). Staffing data were gathered through a ward manager questionnaire.Data were first analysed using systematic reading, familiarisation and open coding, undertaken independently by research team members and then in collaborative data analysis workshops. A preliminary coding frame focused on implementation and mechanisms of impact. All interview data were coded against this frame, the use of constant comparative methods enabling the generation of new categories and the comparison of data in relation to these categories. Narrative data summaries and matrix/charting techniques were then used to facilitate comparison with the NPT framework to test and refine emerging theories of implementation processes. All data from observer and PDN field notes, and from quantitative analyses of staffing data, were then systematically interrogated and compared against these emerging theories, the purpose being to use multiple perspectives to elicit more complex and situated understandings.Ethical approval for the study was granted by the Social Care Research Ethics Committee 14/IEC08/1018.In total, 47 interviews were conducted with ward managers (n=4 people), deputy ward managers (n=2), staff nurses (n=8), healthcare assistants (HCAs) (n=7), senior hospital nurses (n=2) and PDNs (n=2). Thirteen people were interviewed once, two people twice and ten people three times. Eleven people declined further interviews after one interview and two people after two interviews. Ward-based interviewees had worked on their current ward between 2 weeks and 14 years, on average 4 years. Two study days and five action-learning sets were observed in full.Findings illustrate the work that staff needed to do, individually and collectively, to implement CLECC in practice. While many of the individual elements of CLECC were possible to implement during the implementation period, sustaining this work beyond this time was difficult for some ward teams to achieve. The findings that follow explain why this was the case.Interview and observation data clearly indicated that all care staff were able to articulate activities associated with the CLECC intervention. Staff valued the principles behind many concrete CLECC activities, appreciating the focus on staff well-being and consequent impact on patient care quality. For registered nurses (RNs), the CLECC principles resonated with their aspirations for successful team working and patient care. For HCAs, this was a new and welcomed way of thinking about their workplace. Beyond the activities staff were directly involved in, they struggled to visualise the purpose and potential of CLECC. Staff tended to associate CLECC with cluster discussions that took place part way through each shift, thus providing an opportunity to gather as a team and check on each other’s well-being.“So, whereas before they might know that orange bay is heavier than green bay, they might not necessarily have volunteered to go and help. Now they are much more aware that if they are going – well actually we’re struggling – well, we’re not, we’ll come and help you and I think that’s because of the [clusters] and the fact that we’re all sitting down and going – is there anything we can do to help you? And if they are going – well actually I’ve got a really poorly patient, so I’ve been struggling with the others – right – well then – we’ll come and help you. And it’s made them more aware of each other.” N003 (HCA)All staff attended the study days and, on prompting, were able to link these sessions with CLECC. Participating in a study day where only other team members were present was considered unusual and was welcomed. Staff saw the study day as a way of ensuring that they were working together and an opportunity to engage with the ward vision, which was not previously explicit. The most important aspect of the study day was the chance to get to know each other, which staff reported they had not had the opportunity to do previously.“We had the study days and they were all very good and I found that I got to know the different people within those study days, or how they felt and I thought – oh, I didn’t know that. So that was useful.” N001 (Staff Nurse)The ward managers and PDNs charged with facilitating CLECC were involved in a wider range of CLECC activities; these individuals and the senior nurse managers were clearly able to articulate the underlying philosophy of CLECC and to identify associated behaviours.“To me CLECC is about giving staff tools to ensure that they support themselves to do a hard job. So it’s about providing – a nurse with the knowledge of what they need to deliver… compassionate care or high-quality person-centred care, whatever you want to describe it as – every day, at a high quality standard, is what we have to aim for, but also with you having some insight into how your behaviour affects both your patient and your staff.” SN002 (Director)On average over a third of staff left over the course of the study, consistent with other wards we were monitoring, and one senior manager viewed staff turnover as a result of CLECC as a positive outcome:“[CLECC] exposed some practices, provided a culture where people could talk openly about how other members of staff made them feel; there has been a bit of a churn, so maybe some people that needed to go. People have now felt they’ve got a voice and, again, if people aren’t doing what we need them to do, then they need to go.” SN002 (Director)But there was no provision for inducting newly arrived staff into CLECC, limiting their opportunity to make sense of CLECC.In summary, although ward staff appreciated the potential value of CLECC, their understanding of CLECC was limited to and shaped by the concrete activities that they experienced. Additional knowledge about the underpinning principles of CLECC did not filter down to the team as a whole, with no evidence that coherence improved over time from the original induction into CLECC activities.Staff were keen to participate in CLECC, but it was not always clear whose responsibility it was to ensure it happened. Each PDN worked simultaneously with two wards in their allocated hospital, organising specified CLECC activities. Each took a different approach and this influenced the degree of ownership of the intervention by the ward staff. One PDN had an autocratic style of leadership and deliberately undertook to transfer ownership for making things happen, seeing her role as informing teams how things should be done by them:“[The PDN] was very adamant that it wasn’t her responsibility to do cluster, it could have been anybody’s….” N008 (HCA)After the implementation period, staff on those wards reported that cluster discussions were no longer taking place because no one senior was making them happen. The other PDN had a more democratic style of working and actively worked with staff to make CLECC more flexible and fit better with resource pressures.“It [cluster meeting] doesn’t always stick to that time. It kind of depends how it’s going. So we’ve had like busy days when stuff’s been happening on the ward. At one point they [nursing staff] kind of ask permission to make it [cluster meeting] later, it’s kind of sad. But I’m like… ‘yeah, do it whatever time it works in the ward. If we can do it, that’s a bonus’. So quite often it’s the [HCAs] asking for it [cluster meeting].” N035 (PDN)Although this PDN and more senior team members initially originated the cluster discussions, as the intervention became embedded over time others, including HCAs, called them without waiting for more senior initiation. Cluster discussions on these wards continued to run after the implementation period.“They [HCAs] will remind whoever is in charge of the ward, and say ‘Are we having a [cluster] today?’ I’ve seen that quite a few times.” N035 (PDN)CLECC also gave staff, including HCAs, the opportunity to see themselves as innovators, providing a mechanism through which individuals could articulate their ideas for improving practice on the ward. Staff felt more empowered than before to respond to ideas and to implement change, approaching the ward manager and the matron simultaneously, when previously communication was directed through the ward manager.“Quite a few of the staff have got involved in various different things that have come out of the study days, what they wanted to change, and thought they could do better. And they’ve gone off and sort of little groups, or twos and threes, and are bringing that stuff back, passing it through the Matron.” N030 (Ward Manager)Not all ideas were implemented in practice, which appeared to be linked with uncertainty about whose role it was to realise them or to authorise them. Staff clearly had an expectation that the ward manager or matron had the requisite authority to realise the ideas and the lack of ‘follow through’ was demoralising for the staff involved.“Some of them felt a little bit disappointed that they’d made these suggestions and took their time to do them and then no one really followed it through or said – yes, we can use that or no we can’t. It just got left.” N001 (Staff Nurse)Everyone interviewed reflected that they saw CLECC as a way to build the team and improve care, and this ethos underpinned their participation in prescribed activities. Consequently study days and action learning sets were all well attended. Fortnightly CLECC meetings between ward managers and their matrons did not go ahead in one hospital site, indicating a lack of clarity about the role of the matron in making CLECC happen.“Both ward [managers] felt that there has been a negative impact from the lack of support from the matron. Items identified by the nursing teams that were considered areas requiring improvement were unsupported and even in some instances rejected.” N036 (PDN field note)Already established meetings between ward managers and their matron at the other hospital site appeared to be linked with a more proactive matron role in supporting CLECC.“So my matron’s been very supportive the whole way through; we’ve kept in regular contact. She’s been asking for updates, she’s known about the interventions that we’ve done on the ward and has been really supportive.” N034 (Ward Manager)Consequently, while the majority of staff were keen to participate, the extent to which individuals saw it as their role to make CLECC happen varied between professional position and level in the organisation.Whether or not the activities went ahead as planned was mediated by the extent to which the proposed activity harmonised with the priorities of the wider hospital organisation and resources available to the ward team. A particular influence was the organisational priority afforded to material patient care activities over CLECC activities in the context of high patient care workloads. Staff reported struggling to find the time to engage with cluster discussions. The planned 20 min reflective learning sessions proved impossible to integrate into ward practice. CLECC’s flexibility enabled staff to develop strategies that partly overcame these barriers.“Because [the clusters are] 5 min you can work it and actually if you’re having a day where you’re too busy to run them, then that’s the day that you realise that you need to go round and make sure everyone’s okay…And I think that’s definitely been my biggest struggle throughout it all – it’s just being able to release staff to do things.” N005 (Ward Manager)Staff reported that senior hospital managers had endorsed the work that had resulted from the CLECC intervention, suggesting that the benefits were visible and valued outside of the immediate ward team.“They seemed to be really positive about it and [visiting senior manager] said – ‘if this is working for you, continue.’” N009 (HCA)Nonetheless, staff’s participation in CLECC activities was viewed as secondary importance to providing patient care. The degree of support from individual matrons also seemed linked to leadership resources being made available by the organisation to support the team with CLECC:“I assumed that my matron was working with the ward [managers] on a weekly basis but I doubt it was what I expected it to be. So we should have put more nursing leadership resources into it, just to provide that support and recognise it.” SN002 (Director)Many cluster discussions proved possible to integrate into the working day and went ahead during the implementation period but were less readily convened when patient care demands were very high and staffing resource was low. CLECC properties of plasticity enabled staff to develop and adapt practices that suited local circumstances but were constrained by the available resources and priorities of the wider organisation.Staff reported benefits to personal well-being and capacity to care from CLECC participation. They spoke of engaging more consciously and deliberately with patients as people, prioritising this over the completion of tasks. Staff reported that their practice was already compassionate, but CLECC had given them opportunities to value these practices and to make further commitment to compassion.“CLECC, for me, is about giving the staff the empowerment to feel like they can sit and do things with patients that are compassionate rather than task orientated, so rather than just doing the [observations] and just doing the washes, just having a chat with the patient about their life, their family or sitting and doing an activity with them; rather than just, we’ve got to get the washes done, we’ve got to get the observations done – which do still need to be done but it’s about giving the staff that that empowerment of being able to say, let’s do something a bit different.” N034 (Ward Manager)CLECC was associated with an improvement in staff morale and staff well-being and viewed as impacting positively on patient care. Interestingly, some of the legitimacy for CLECC practices seemed to come from the fact that they were part of the CLECC intervention and perhaps a research study. One interviewee cited an instance in which a senior manager visiting the ward came across a cluster discussion, which were also used by some teams to make sure that staff had a drink of water.“I don’t know who it was, but someone very high in the hospital [came to the ward] and was like, ‘why are people standing and drinking on the corridor?’” N025 (HCA)Once the manager was told the cluster discussion was part of CLECC, she was reported to have then understood the purpose behind an activity considered to be unusual enough to be remarked on.The improved team working reduced the burden for some staff and provided opportunities to undertake activities that previously would have been rare occurrences.“But because of the task orientated work – we’ve managed to go, right, we’ve finished, [they] haven’t and then so we can go, right, we’ll give you guys a hand and then we can all be finished together. And then that means we’ve got more time to do things that we might not be able to normally do, like – wash someone’s hair, do their nails.” N009 (HCA)The principles that underpin CLECC appeared to be well embedded into the teams, even several months after the end of the implementation period. Some of the wards continued with the cluster discussions, and even on wards where they had not continued, staff valued them as an indicator of supporting each other and working together. Attention to supporting each other appeared to have increased the relational capacity of individual team members and the team as a whole.This study aimed to identify and explain the extent to which the planned CLECC intervention was implemented by nursing staff into existing work practices, to enable conclusions to be drawn about how interventions of this kind can be optimised and sustained to support compassionate care in acute settings.While CLECC had limited coherence for some staff, it was welcomed by teams and served as a broader stimulus to collective action. CLECC developed cultures in which reflection, learning, mutual support and innovation were legitimised within the work-team, and in which expertise was seen to be distributed more widely between managers, RNs and HCAs. CLECC moved all the teams further along the continuum to becoming more expansive learning environments,Staff at all levels were able to identify the benefits to patient care of ward staff engaging in CLECC activities, echoing other findings that the creation of unmanaged spaces for work-team members to ‘take shelter’ provides the potential for valued learning and social support for difficult work with clients.Factors outside of the direct influence of the ward teams mediated the impact and sustainability of the intervention, in particular the institutional norms that legitimated staff’s participation (or not) in CLECC activities and the interpretation by more senior figures  of what CLECC was and their role in supporting it. While CLECC draws on principles of democratic working, and we saw how HCAs in particular were enabled to take a lead in some CLECC activities, its longer term success relied on cognitive participation from more senior members of the hierarchy. The authority that ward staff had to control how they spent their time, to innovate and to afford their own and colleagues’ well-being some priority varied between teams and was dependent on the signals, or ‘invitational qualities’Our findings indicate that higher and more sustained impact for interventions such as CLECC may only be possible through more substantial restructuring that reshapes the conditions in which people are able to act.These findings reflect that relational work in caregiving organisations depends on individual caregiver agency and on whether or not this work is adequately supported by features of the wider system. Relational capacity may thus be regarded, not just as a property of individual practitioners, but a modifiable and situated property of work-teams. The limitation that institutional norms of legitimate nursing work placed on staff finding time to meet together raises the prospect of lack of relational capacity at a wider system level, and suggests that wider restructuring beyond middle manager roles may in fact be needed to effect substantial and sustained change.


Intricate mechanisms relate cell death processes with immune responses, which have implications for degenerative and inflammatory conditions. We review the therapeutic value of MSCs in terms of preventing regulated cell death (RCD). When cells identify an insult, specific intracellular pathways are elicited for execution of RCD processes, such as apoptosis, necroptosis, and pyroptosis. To some extent, exacerbated RCD can provoke an intense inflammatory response and vice versa. Emerging studies are focusing on the molecular mechanisms deployed by MSCs to ameliorate the survival, bioenergetics, and functions of unfit immune or nonimmune cells. Given these aspects, and in light of MSC actions in modulating cell death processes, we suggest the use of novel functional in vitro assays to ensure the potency of MSCs for preventing RCD. Such analyses should be associated with existing functional assays measuring the anti‐inflammatory capabilities of MSCs in vitro. MSCs selected on the basis of two in vitro functional criteria  could possess optimal therapeutic efficacy in vivo. In addition, we underline the implications of these perspectives in clinical studies of MSC therapy, with particular focus on acute respiratory distress syndrome. S Most studies of mesenchymal stem  cells (MSCs) focus on their anti‐inflammatory, trophic and differentiation abilities, but their ability to prevent regulated cell death (RCD) remains undefined. However, this last function could explain both the regenerative and anti‐inflammatory therapeutic effect of MSCs observed in preclinical and clinical studies. The present report reviews the role of MSCs in preventing RCD, with implications for enhancing their therapeutic efficacy in the clinic. Development of in vitro assays to assess MSC functional potency in preventing RCD is suggested and criteria for selecting MSCs for therapeutic use are proposed. Furthermore, in vivo biomarkers of RCD that can be used for prompt evaluation of the therapeutic effects of MSCs are suggested.Mesenchymal stem  cells (MSCs), in humans, are principally derived from bone marrow and adipose tissues in adults and in neonatal tissues from umbilical cord blood and placenta , 2, 3. RToday, MSCs are under intense clinical investigation for regenerative medicine because of their differentiation and trophic abilities , 13, 14 Substantial efforts have been undertaken by the translational community to standardize methods for producing, selecting, and using MSCs in the clinic , 6. NotaEmerging evidences indicate a critical role for regulated cell death (RCD) in the pathogenesis of various diseases . By defiThe RCD processes differ by their molecular triggers, molecular pathways engaged and mode of execution . ApoptosNecroptosis is mediated by a mechanism that depends on receptor‐interacting protein kinase 1/3 and mixed lineage kinase‐like protein, whereas pyroptosis is executed in cells by a mechanism involving caspase‐1/4/5 and gasdermin D , 26, 27.MSCs are known to improve cell survival and prevent apoptosis, necroptosis and pyroptosis , 2 occurTherefore, innovative therapeutic interventions should simultaneously target RCD and inflammation to optimize cure . The abuThe antiapoptotic properties of MSCs toward immune and nonimmune cells have been demonstrated in some contexts , 45, 46.Therefore, we suggest that to optimize the efficiency of MSC therapy, the ability of MSCs to prevent RCD should be evaluated by in vitro functional assays before the cells are used in clinical interventions . The assBecause the therapeutic effects of MSCs often result from multiple pathways, with or without redundant actions , the in Indeed, RCD represents a therapeutic target for attenuating both tissue damage and inflammation in various disorders  such as A study by Leblanc and colleagues  showed iImprovements in patients with ARDS who received adoptive transfer of MSCs were associated with a rapid decrease in levels of markers of cell death . SignifiThus, RCD biomarkers could be measured to monitor and rapidly predict the outcomes of a given MSC treatment in patients with ARDS. This analysis is crucial to readily evaluate the response of the intervention in patients and could be used to adapt and appropriately improve the treatment. Leblanc and colleagues suggested that MSCs have therapeutic efficacy for ARDS . FurtherMSCs are remarkable from therapeutic perspectives, given the ease with which we can obtain a significant number of genetically stable MSCs and the number of diseases that can be treated because of the intrinsic properties of MSCs . Today, A.N., conception and design, figure/table design, manuscript writing, final approval of manuscript; N.S., N.E., K‐i.Y., and N.B.: manuscript writing, final approval of manuscript; L.S. and F.D.: conception and design, manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.


Basic skills training in laparoscopic high-fidelity simulators (LHFS) improves laparoscopic skills. However, since LHFS are expensive, their availability is limited. The aim of this study was to assess whether automated video analysis of low-cost BlackBox laparoscopic training could provide an alternative to LHFS in basic skills training.Medical students volunteered to participate during their surgical semester at the Karolinska University Hospital. After written informed consent, they performed two laparoscopic tasks (PEG-transfer and precision-cutting) on a BlackBox trainer. All tasks were videotaped and sent to MPLSC for automated video analysis, generating two parameters (Pl and Prtcl_tot) that assess the total motion activity. The students then carried out final tests on the MIST-VR simulator. This study was a European collaboration among two simulation centers, located in Sweden and Greece, within the framework of ACS-AEI.P < 0.0001; 0.34, P = 0.0009; 0.45, P < 0.0001, respectively) as well as to the Prtcl_tot variable in that same exercise . However, the correlations were exclusively shown in the group with less PC gaming experience as well as in the female group.31 students , mean age of 26.2 ± 0.8 years, participated in the study. However, since two of the students completed only one of the three MIST-VR tasks, they were excluded. The three MIST-VR scores showed significant positive correlations to both the Pl variable in the automated video analysis of the PEG-transfer (RSquare 0.48, Automated video analysis provides accurate results in line with those of the validated MIST-VR. We believe that a more frequent use of automated video analysis could provide an extended value to cost-efficient laparoscopic BlackBox training. However, since there are gender-specific as well as PC gaming experience differences, this should be taken in account regarding the value of automated video analysis. Laparoscopic simulation training to proficiency levels has been shown to increase both basic skills –5 and thBasic skills training in BlackBoxes within a structured curriculum is a less costly alternative to LHFS-simulation training and has been shown to provide improvement of both basic as well as laparoscopic technical skills , 12. HowVarious motion tracking systems have been proposed in the literature to address issues relevant to subjective and manual evaluation of surgical performance . Other aThere is some evidence of a positive relationship between video gaming experience and the acquisition of laparoscopic simulator surgical skills. However, the results are conflicting as there is a lack of a standardized scoring system and, therefore, further research on this subject is advised –24. GlasThe aim of this study was to analyze if automated video analysis of basic skills training performance could be a valid method of increasing the value of BlackBox training and if it, in part, could provide an alternative to high-fidelity skills training in simulators, and furthermore to analyze if there were gender- or PC gaming experience-specific differences in the efficacy of BlackBox training. This study was a European collaboration within the framework of ACS-AEI.Query.Thirty-one medical students  with a mean age of 26.2 ± 0.8 (Mean ± SEM) were recruited for this study during their surgical semester at the Karolinska University Hospital. The study was conducted at the Center for Advanced Medical Simulation and Training (CAMST), Karolinska Institutet, Stockholm Sweden and the video analysis was carried out at MPLSC, Athens University Medical School, Athens, Greece.Students were recruited on a voluntary basis, when the study was presented to them, at the beginning of their surgical semester. After signing informed consent forms, the recruited students performed two laparoscopic tasks (PEG-transfer and cutting a circular gauze) on a BlackBox  Fig. . PEG-traOral instructions were given to each student before the study. None of the students had any prior surgical or laparoscopic experience, nor did they have any prior training before the task. However, some of the students had prior simulation training experience. The BlackBox is a wooden box with three holes located on the top, one for a camera and two for the laparoscopic instruments Fig. . The camThe goal of the first task was to move a series of six rubber rings located on one side of the pegboard, to the other side of the pegboard by the use of graspers, with the non-dominant hand moving the rubber rings to the dominant one before placing them on the other side of the pegboard. This process was then reversed by switching direction and hands to bring back the rubber rings to their starting position Fig. . The goaPeer to Peer, comparing subjects within one class, and Progression, used to show a subject’s performance over a specified set of examinations of the same configurations  were excluded.Each video was analyzed in a blind fashion to assess the total movement in the video based on optical flow metrics. In particular, the feature detection method  was applBased on the aforementioned analysis, the following two metrics were derived from each video: ‘Pl’ the total displacement (‘path-length’) of all particles, and ‘Prtcl_tot’ the total number of particles detected, across all frames. No user input or training of the algorithm was required prior to the analysis. The only input was the video file, and the output was the two previous metrics.t test and the results given as Mean ± SEM. A P-value <0.05 was regarded as statistically significant. Statistical analyses were carried out using JMP® version 12.1.0 (64-bit) (SAS Institute Inc).The results of the automated video analysis regarding ‘Pl’ and ‘Prtcl_tot’ were correlated to the results of the three MIST-VR scores using linear fit. The results are given as RSquare. Differences in MIST-VR scores between females and males as well as between infrequent and frequent PC gamers in the PEG-transfer exercises were analyzed using Student’s P < 0.0001; 0.34, P = 0.0009; 0.45, P < 0.0001) , whereas Prtcl_tot only showed a significant correlation in the first two MIST-scores , whereas two only performed one MIST-VR task and thus were excluded , whereas there were no correlations in the male group , whereas no correlations were found in the male group.Surprisingly, there were gender-specific differences between Pl of the PEG-transfer group and the MIST-VR scores since correlations were only found in the female group , in contrast to the frequent PC gaming group.Furthermore, in the PEG-transfer exercise we noted significant and even more pronounced correlations between the Pl variable in the video analysis and the MIST-VR scores  in the Although the more experienced PC gamers were predominantly males (67%), there were no significant differences in simulation performance as assessed by the MIST-simulation scores neither between males and females nor between experienced or not experienced PC gamers. We, however, noted a training effect of the simulation training since the MIST-VR scores decreased in all groups between the first and last MIST-VR simulation (low scores indicating better performance) Table .Table 2MThis study shows a strong correlation between the results of the automated video analysis of the two consecutive laparoscopic tasks on a BlackBox trainer and the results of the three MIST-VR-task procedures. Furthermore, this study could also identify that the correlations between the results of the automated video analysis and the MIST-VR results were gender specific and correlated to computer gaming experience.The findings of this study in relation to gender-specific differences regarding computer gaming experience are in accordance with other studies carried out , 32. HowOne interesting finding of the present study was that the strong correlations between automated video analysis and the performance, as assessed by the MIST-VR simulation tests, were only found in the subgroups of females and non-experienced PC gamers. These findings may indicate that the use of automated video analysis, as described in this study and elsewhere , 21, 34,Our finding of a training effect of the laparoscopic MIST-VR simulator exercises is in accordance with other studies , 5, 36. One of the main advantages of the MIST-simulator is that it provides an instant feedback and that the exercises regarding instrument handling are similar to laparoscopic cholecystectomies although the graphics are outdated. One of the drawbacks of the automated video analysis is that the parameters measured here capture the total activity of moving objects in the video, and are not specific to the tool movement. Nevertheless, the total motion activity in the training videos is mostly due to the movement of the tools, and less to other moving objects . Moreover, in its present form , video aHowever, our model is a low-cost alternative to more advanced VR simulators that are not always available on a large scale in most countries. The financial cost of our system is in the range of US $150 and a mass production could decrease it substantially.In conclusion, we provide evidence that in subgroups of novices there are strong correlations between our automated video analysis of BlackBox training and performance in a validated laparoscopic simulator. One potential use of our model is to assess basic skills training in a low-cost way, thus leading to enhancement of surgical performance as well as to evaluate subjects with various levels of experience.