Differential Status Research Articles

Seeing the Whole Together Through Relational Mapping: A Method for Engaging in Complex Systems Change

Complex global challenges cut across organizational and sectoral boundaries, increasing calls for holistic responses. Seeing the whole across diverse stakeholders is challenging for participants in complex systems, yet doing so is essential for engaging effectively in systems change. A promising new method known as relational mapping, based on relational coordination theory, has been used to facilitate complex systems change. Here, we propose that relational mapping can be applied to help participants (1) see the whole in relation to the parts, (2) share their explicit and tacit knowledge, (3) engage in dialogue across distinct perspectives supported by the map as a boundary object, (4) reduce the impact of power differences, and (5) prepare to co-design interventions from multiple perspectives. This paper presents an illustrative case of relational mapping in the context of public administration in a South Carolina community engaging in complex systems change. Our analysis suggests a set of boundary conditions for the success of relational mapping, including the willingness of participants to seek integrative solutions to a conflict or problem and facilitation that is attentive to differential power and status among stakeholders. We conclude that relational mapping is a powerful, theoretically grounded method that enables the visual assessment of multi-level stakeholder relationships, preparing participants to engage in complex systems change and then present a template for use of relational mapping more broadly.

A temporal (phospho-)proteomic dataset of neurotrophic receptor tyrosine kinase signalling in neuroblastoma

Neurotrophic receptor tyrosine kinases (TrkA, TrkB, TrkC), despite their homology, contribute to the clinical heterogeneity of the childhood cancer neuroblastoma. TrkA expression is associated with low-stage disease and is often seen with spontaneous tumour regression. Conversely, TrkB is present in unfavourable neuroblastomas that often harbour amplification of the MYCN oncogene. The role of TrkC is less clearly defined, although some studies suggest its association with a favourable outcome. Understanding the differences in activity of Trk receptors that drive divergent clinical phenotypes as well as the influence of MYCN amplification on downstream Trk receptor signalling remains poorly understood. Here, we present a comprehensive label-free mass spectrometry-based total proteomics and phosphoproteomics dataset (432 raw files with FragPipe search outputs; available on PRIDE with accession number PXD054441) where we identified and quantified 4,907 proteins, 16,744 phosphosites and 5,084 phosphoproteins, derived from NGF/BDNF/NT-3 treated TrkA/B/C-overexpressing neuroblastoma cells with differential MYCN status. Analysing our dataset offers valuable insights into TrkA/B/C receptor signalling in neuroblastoma and its modulation by MYCN status; and holds potential for advancing therapeutic strategies in this challenging childhood cancer.

Correlation of bone age development with overweight and obesity in 23,305 children from Beijing.

The aim of this study was to observe the influence of differential nutritional status on bone age (BA) change according to body mass index (BMI) and analyze the risk of advanced bone age in children with overweight and obesity. In total 23,305 children from Beijing were included in this cross-sectional study. Childhood overweight and obesity were defined according to the China and World Health Organization growth criteria. The data were analyzed by the R coding platform version 4.3.0. Under the Chinese criteria, 29%, 15%, and 4% of boys with overweight; 33%, 33%, and 3% of boys with obesity; 39%, 25%, and 2% of girls with overweight; and 37%, 42% and 1% of girls with obesity had advanced, significantly advanced and delayed BA, respectively. After adjustment, overweight (odds ratio, 95% confidence interval, P under the Chinese criteria: 2.52, 2.30-2.75, <0.001 and 4.54, 4.06-5.09, <0.001) and obesity (4.31, 3.85-4.82, <0.001 and 14.01, 12.39-15.85, <0.001) were risk factors for both advanced BA and significantly advanced BA. Different nutritional statuses lead to differences in children's BA development. Children with overweight and obesity have higher rates of advanced BA under two growth criteria, and girls have more advances in BA than boys do. Overweight and obesity are risk factors for advanced BA.

Unveiling the role of protein kinase A (PKA) activity in bovine oviductal epithelial cells: implications on apoptotic signaling pathways during the estrous cycle.

The complex interactome crucial for successful pregnancy is constituted by the intricate network of endocrine and paracrine signaling pathways, involving gametes, embryos, and the female reproductive tract. Specifically, the oviduct exhibits distinct responses to gametes and early embryos during particular phases of the estrus cycle, a process tightly regulated by reproductive hormones. Moreover, these hormones play a pivotal role in orchestrating cyclical changes within oviductal epithelial cells. To unravel the molecular mechanisms underlying these dynamic changes, our study aimed to investigate the involvement of protein kinase A (PKA) in oviductal epithelial cells throughout the estrus cycle and in advanced pregnancy, extending our studies to oviductal epithelial cell in primary culture. By a combination of 2D-gel electrophoresis, Western blotting, and mass spectrometry, we identified 17 proteins exhibiting differential phosphorylation status mediated by PKA. Among these proteins, we successfully validated the phosphorylation status of heat shock 70kDa protein (HSP70), aconitase 2 (ACO2), and lamin B1 (LMNB1). Our findings unequivocally demonstrate the dynamic regulation of PKA throughout the estrus cycle in oviductal epithelial cells. Also, analysis by bioinformatics tools suggest its pivotal role in mediating cyclical changes possibly through modulation of apoptotic pathways. This research sheds light on the intricate molecular mechanisms underlying reproductive processes, with implications for understanding fertility and reproductive health.

O-028 DNA methylation testing with cervical secretion can serve as a non-invasive biomarker for predicting successful implantation in frozen-thawed embryo transfer

Abstract Study question Is cervical secretion gene methylation profile associated with embryo implantation outcome, and can be used as a non-invasive biomarker for pregnancy prediction in FET cycles? Summary answer Cervical secretion gene methylation profiles show significant associations with implantation outcomes, and can serve as a non-invasive predictor of successful pregnancy in FET cycles. What is known already Cause of implantation failure remains a black box in reproductive medicine, and the exact mechanism of how endometrial receptivity is still behind the curtain. Epigenetic modifications including DNA methylation regulate gene expression and may alter endometrium receptivity. Endometrial DNA methylation changes vigorously only during the reconstruction of a new endometrium throughout each menstrual cycle, suggesting a unique methylation landscape in each regenerated endometrium. Cervical secretions contain implantation-related cytokines and genetic material from endometrium, and the DNA methylation profiles of cervical secretions is highly correlated with endometrium. Therefore, cervical secretions can be used as non-invasive surrogates for investigating endometrial condition. Study design, size, duration This multi-centered, case-control study enrolled 72 infertile females who entered the FET cycle with at least one high-quality blastocyst available between Aug 2017 and Dec 2022. Embryos assessed by Gardner scoring system with grade 3BB or above were defined as high-quality. Cervical secretions were collected on the day right before embryo transfer, and then DNA methylation profile were assessed. Participants/materials, setting, methods These females aged between 20-45 years-old with regular menstruation cycle (24-35 days) and normal BMI (17.5–32.0 kg/m2). Uterine cavity lesions and hydrosalpinx without treatment, adenomyosis, large uterine myoma that distorted endometrium, and thin endometrium (&amp;lt;7mm) were excluded. The association between the cervical secretion gene methylation profile and pregnancy outcome was evaluated via quantitative methylation-specific PCR (qMSP), methylation array, and genome-wide analysis. Different machine learning algorithms were utilized for differential methylation analysis and implantation prediction. Main results and the role of chance Cervical secretion DNA methylome differed between receptive (pregnant) and non-receptive (non-pregnant) FET cycle with genome-wide methylation array. The accuracy of pregnancy outcome prediction using top 2000 differential methylation probes (DMPs) in promoter region can be as high as 86% in all tested sample and 96.4% in pregnant samples. Bioinformatic analysis of six selected candidate genes related to implantation from literature review showed significant differences in DNA methylation between receptive and non-receptive endometrium. The methylation level of the six candidate genes with eight regions were measured by the qMSP platform and showed varying degrees of differential methylation status between the pregnancy and non-pregnancy groups. The AUCs for a single gene/region ranged from 0.53–0.67 for implantation prediction. Ten different machine learning models were applied and the results showed that the combination of candidate genes’ DNA methylation profiles could differentiate pregnant from non-pregnant samples with an accuracy as high as 86.67% and an AUC of 0.81 by logistic regression model. The best model from different machine learning algorithms using selected DMPs of genes related to window of implantation (WOI) can predict ongoing pregnancy with accuracy rates as high as 86.78% and AUC values up to 0.91. Limitations, reasons for caution The results obtained from a relatively small cohort size, and the timing of sample collection is on the day right before embryo transfer. Further investigation of methylation profiles in late follicular phase along with large sample size with euploid embryo transfer are mandatory for validation and clinical applications. Wider implications of the findings We firstly demonstrated the feasibility of cervical secretion methylation profile as a non-invasive testing for pregnancy prediction. The promising result may contribute to predicting endometrial receptivity for cycles with a favorable endometrium and maximize the successful pregnancy rate of embryo transfer. Trial registration number not applicable

Single-cell mitochondrial sequencing reveals low-frequency mitochondrial mutations in naturally aging mice.

Mitochondria play a crucial role in numerous biological processes; however, limited methods and research have focused on revealing mitochondrial heterogeneity at the single-cell level. In this study, we optimized the DNBelab C4 single-cell ATAC (assay for transposase-accessible chromatin) sequencing workflow for single-cell mitochondrial sequencing (C4_mtscATAC-seq). We validated the effectiveness of our C4_mtscATAC-seq protocol by sequencing the HEK-293T cell line with two biological replicates, successfully capturing both mitochondrial content (~68% of total sequencing data) and open chromatin status simultaneously. Subsequently, we applied C4_mtscATAC-seq to investigate two mouse tissues, spleen and bone marrow, obtained from two mice aged 2 months and two mice aged 23 months. Our findings revealed higher mitochondrial DNA (mtDNA) content in young tissues compared to more variable mitochondrial content in aged tissues, consistent with higher activity scores of nuclear genes associated with mitochondrial replication and transcription in young tissues. We detected a total of 22, 15, and 21 mtDNA mutations in the young spleen, aged spleen, and bone marrow, respectively, with most variant allele frequencies (VAF) below 1%. Moreover, we observed a higher number of mtDNA mutations with higher VAF in aged tissues compared to young tissues. Importantly, we identified three mtDNA variations (m.9821A>T, m.15219T>C, and m.15984C>T) with the highest VAF in both aged spleen and aged bone marrow. By comparing cells with and without these mtDNA variations, we analyzed differential open chromatin status to identify potential genes associated with these mtDNA variations, including transcription factors such as KLF15 and NRF1. Our study presents an alternative single-cell mitochondrial sequencing method and provides crude insights into age-related single-cell mitochondrial variations.

High hyperdiploid karyotype with ≥ 49 chromosomes represents a heterogeneous subgroup of acute myeloid leukemia with differential TP53 mutation status and prognosis: a single-center study from China.

High hyperdiploid karyotype with ≥ 49 chromosomes (which will be referred to as HHK) is rare in acute myeloid leukemia (AML). The European leukemia network (ELN) excluded those harboring only numerical changes (with ≥ 3 chromosome gains) from CK and listed them in the intermediate risk group, while the UK National Cancer Research Institute Adult Leukaemia Working Group classification defined ≥ 4 unrelated chromosome abnormalities as the cutoff for a poorer prognosis. Controversies occurred among studies on the clinical outcome of HHK AML, and their molecular characteristics remained unstudied. We identified 1.31% (133/10,131) HHK cases within our center, among which 48 cases only had numerical changes (NUM), 42 had ELN defined adverse abnormalities (ADV) and 43 had other structural abnormalities (STR). Our study demonstrated that: (1) No statistical significance for overall survival (OS) was observed among three cytogenetic subgroups (NUM, STR and ADV) and HHK AML should be assigned to the adverse cytogenetic risk group. (2) The OS was significantly worse in HHK AML with ≥ 51 chromosomes compared with those with 49-50 chromosomes. (3) The clinical characteristics were similar between NUM and STR group compared to ADV group. The former two groups had higher white blood cell counts and blasts, lower platelet counts, and mutations associated with signaling, while the ADV group exhibited older age, higher chromosome counts, higher percentage of myelodysplastic syndrome (MDS) history, and a dominant TP53 mutation.

Unravelling the oxygen factor - An investigation of transcriptional activation of hypoxia associated placental angiogenesis in recurrent pregnancy loss (RPL) patients from Assam, India

IntroductionRecurrent pregnancy loss (RPL) is defined as the spontaneous loss of two or more consecutive pregnancies before 20 weeks of gestation, and affects 7.46 % of the Indian population. About 40–50 % of RPL cases are idiopathic making it a therapeutic challenge for clinicians. This study focuses on elucidating the role of hypoxia-associated placental angiogenesis in these idiopathic RPL cases. MethodsWhole blood and product of conception (POCs) were collected from RPL patients (N = 87) and cases of voluntary abortions (medically terminated pregnancy, MTP; n = 110) as controls with informed consent. Serum separated from whole blood was used to study the ROS-antioxidant status in the cases and controls through colorimetric assays and ELISA. The mRNA extracted from placental tissue samples were used to determine the hypoxic and angiogenic status in cases and controls through real time PCR. Statistical analysis was also carried out to correlate the differential hypoxic status between RPL and MTP cohorts with the expression of angiogenic factors (VEGFA, VEGFR1 and VEGFR2). ResultsHIF1α mRNA expression was found to be upregulated in the RPL cases. While the serum levels of H2O2 (p = 0.012), guanine oxides and lipid hydroperoxides (LPO) were increased in the RPL cases, reduced glutathione (GSH) was found to be significantly decreased (p = 0.012). Additionally, AUROC analysis also shows an excellent discriminatory ability of 0.850 for serum H2O2 levels. VEGF-A and VEGF-R1 mRNA expression was also found to be downregulated in the RPL cases compared to MTP. DiscussionThis study indicates that increased oxidative stress may lead to aberrations in the VEGF pathway resulting in improper placentation in RPL cases, and subsequently, pregnancy loss.

Identification of colon adenocarcinoma necroptosis subtypes and tumor antigens for the development of mRNA vaccines

BackgroundColon adenocarcinoma (COAD) is a serious public health issue due to high incidence and mortality rate. This study aimed to identify possible tumor antigens and necroptosis subtypes of COAD for the development of mRNA vaccines and the selection of appropriate patients for precision therapy. MethodsGene expression profiles and clinical information for COAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus, respectively. We comprehensively studied the alterations in necroptosis-related genes (NRGs) using cBioPortal, and screened the hub NRGs associated with the prognosis of patients with COAD using Gene Expression Profiling Interactive Analysis 2. Consensuses clustering analysis was performed to identify necroptosis subtypes. Weighted gene co-expression network analysis (WGCNA) was used to identify the co-expression modules of the NRGs. The necroptosis landscape of COAD was assessed using graph learning-based dimensionality reduction. Finally, a drug sensitivity analysis of the two necroptosis subtypes was performed. FindingsTwo tumor antigens, BLC-2-associated X protein (BAX) and interleukin 1 beta (IL1B) were identified based on their associations with prognosis of patients and antigen presenting cell infiltration. Two necroptosis subtypes (N1 and N2) were distinguished in patients with COAD, and they were characterized by their differential survival status and molecular expression levels of immune checkpoint proteins and immunogenetic cell death modulators. Furthermore, the necroptosis landscape of COAD indicated that individual patients had obvious heterogeneity. Co-expression modules were identified using WGCNA, and the hub NRGs were found to be involved in various immune processes. Drug sensitivity analysis indicated that there were significant differences in drug sensitivity between the N1 and N2 subtypes. Cell experiments suggested that both overexpression of BAX and IL1B promoted necroptosis of COAD cells and enhanced the cytotoxicity of CD8+ T cells. InterpretationBAX and IL1B are potential antigens for the development of anti-COAD mRNA vaccines, specifically for patients with the N2 subtype. Consequently, this study will guide the development of more effective immunotherapeutic approaches and the identification of appropriate patients.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

Genome-Wide Methylation Analysis in Two Wild-Type Non-Small Cell Lung Cancer Subgroups with Negative and High PD-L1 Expression.

We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in mutation analysis and had either high (TPS > 50%; n = 10) or negative (TPS < 1%; n = 10) PD-L1 status to be integrated into our study. To examine the methylation of 866,895 specific sites, we utilized the Illumina Infinium EPIC bead chip array. Both hypermethylation and hypomethylation play significant roles in tumor development, progression, and metastasis. They also impact the formation of the tumor microenvironment, which plays a decisive role in tumor differentiation, epigenetics, dissemination, and immune evasion. The gained methylation patterns were correlated with PD-L1 expression. Our analysis has identified distinct methylation patterns in lung adenocarcinomas with high and negative PD-L1 expression. After analyzing the correlation between the methylation results of genes and promoters with their pathobiology, we found that tumors with high expression of PD-L1 tend to exhibit oncogenic effects through hypermethylation. On the other hand, tumors with negative PD-L1 expression show loss of their suppressor functions through hypomethylation. The suppressor functions of hypermethylated genes and promoters are ineffective compared to simultaneously activated dominant oncogenic mechanisms. The tumor microenvironment supports tumor growth in both groups.

Abstract 3034: Krüppel-like factors in regulation of gastric cancer progression

Abstract Introduction: Gastric cancer (GC) is the third leading cause of cancer-associated mortality and the fifth most frequently diagnosed cancer worldwide. The development and progression of GC is a complicated multistage process subjected to dynamic changes in gene regulation. Investigating the effects of these changes is therefore critical for improving our knowledge about GC initiation and progression. Krüppel-like factors (KLFs) are evolutionarily conserved zinc finger-containing transcription factors with altered expressions and diverse regulatory functions in GC. We previously reported that there is loss of KLF4 in GC. Here we report that KLF4 is a crucial regulator of KLF5 expression and vice versa and together they regulate GC cell behavior. Methods: The Cancer Genome Atlas (TCGA) and pan cancer data of differential expressions of KLFs in GC was analyzed. TCGA RNAseq and clinical data for the differential expression status of KLF genes in normal and cancerous tissues and the clinicopathological association was accessed from the UALCAN web portal. The expressions of KLF4 and KLF5 in human normal stomach and GC and orthotopic xenografts were detected by immunohistochemistry (IHC). KLF4, KLF5 cross regulation was evaluated by gene manipulation of each gene individually and checking the expression of the other factor in GC cells. The effect of KLFs on GC cell proliferation, migration, and invasion was determined by Ki67 staining of tissues, PrestoBlue cell viability assay, scratch wound and transwell cell invasion assays. Results and Conclusion: Our results indicated that not only KLF4 and KLF5 have contrasting expressions and effects in GC but also these two factors cross regulate each other. While lower expressions of KLF4 was found in human GC and orthotopic xenografts, increased expression of KLF5 was found in both human GC tissue and orthotopic xenografts. KLF4 silencing and CRISPR/Cas9 knockdown in human GC cells resulted in increased expression of KLF5 which was associated with increased GC cell proliferation and invasiveness. Interestingly, KLF5 knockdown by CRISPR/Cas9 in human GC cells on the other hand resulted in increased expression of KLF4, which was associated with decreased GC cell growth. Our data therefore suggests that the differential expressions of KLF4 and KLF5 might serve as valuable prognostic markers in GC. Citation Format: Sooraj Kakkat, Prabhat Suman, Sandeep Goswami, Sujit Basu, Martin J. Heslin, Elba A. Turbat-Herrera, Veronica Ramirez Alcantara, Chandrani Sarkar, Debanjan Chakroborty. Krüppel-like factors in regulation of gastric cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3034.

Differential Status of Serum Arginine, Arginase and Nitric Oxide in Patients of Chronic and Advanced Stage Kidney Disease Undergoing Hemodialysis

Background: Chronic kidney disease (CKD) is characterized by deterioration of endothelial function which is associated with reduced availability of nitric oxide. The objective of the study was to assess the differential status of the serum levels of arginine, arginase, NO, urea and creatinine in CKD patients not on hemodialysis and in end stage renal disease (ESRD) patients receiving hemodialysis. Material and Methods: In this case control study, clinically diagnosed 30 CKD patients (group I), 30 ESRD patients before hemodialysis (Group II), 30 patients with ESRD after first hemodialysis (group III) and 30 patients with ESRD after second hemodialysis (Group IV) were included. 30 healthy volunteers were included for comparison. Serum arginine, arginase, nitric oxide, urea and creatinine were estimated by colorimetric and spectrophotometric methods. Results: Serum creatinine and urea levels were evaluated to determine the severity of renal dysfunction. A significantly decreased serum arginine and nitric oxide levels whereas significantly increased serum arginase levels were observed in ESRD patients when compared to levels in CKD patients. Serum creatinine levels were significantly decreased after second hemodialysis. But there was no significant change in the serum levels of arginine, arginase and nitric oxide in ESRD patients after first and second hemodialysis compared to pre-dialysis group. Conclusion: The findings of the study throw light upon the differential status of serum arginine, arginase and nitric oxide in CKD and in ESRD patients. The evaluation of decreased nitric oxide levels coupled with elevated arginase activity may help in assessing progression of CKD to ESRD along with traditional markers of kidney function. Additionally, evaluation of serum arginase activity may provide useful prognostic information, with large study group and further follow-up, in hemodialysis patients.

Evaluation of Weed Species for Host Status to the Root-Knot Nematodes Meloidogyne enterolobii and M. incognita Race 4.

Weeds that compete with valuable crops can also host plant-parasitic nematodes, acting as a source of nematode inoculum in a field and further damaging crops. The host status of 10 weed species commonly found in North Carolina, USA, was determined for the root-knot nematodes Meloidogyne enterolobii and M. incognita race 4 in the greenhouse. Each weed species was challenged with 5,000 eggs/plant of either M. enterolobii or M. incognita race 4, with five replicate plants per treatment in two separate greenhouse trials. Root galling severity and total number of nematode eggs per root system were recorded 60 days after inoculation. Reproduction factor (Rf = final nematode population/initial nematode population) was calculated to determine the host status of each weed species to M. enterolobii and M. incognita race 4. Four weed species (Datura stramonium, Digitaria sanguinalis, Senna obtusifolia, and Cyperus esculentus) were poor hosts (Rf < 1) to both nematode species, and roots of these weed plants did not display galling. Four weed species (Ipomoea hederacea, Amaranthus palmeri, Portulaca pilosa, and Ipomoea lacunosa) were hosts (Rf > 1) to both nematode species, and all had observable root gall formation. Sida rhombifolia and Cyperus rotundus were poor hosts to M. enterolobii but susceptible hosts to M. incognita. This study documents a differential host status of some common weeds to M. enterolobii and M. incognita race 4, and these results highlight the necessity of managing root-knot nematodes through controlling weeds in order to protect valuable crops.

A Longitudinal Study on Campylobacter in Conventionally Reared Commercial Broiler Flocks in the United States: Prevalence and Genetic Diversity.

Poultry meat contaminated with Campylobacter, a major bacterial cause of foodborne gastroenteritis worldwide, is considered the primary source of human campylobacteriosis. Thus, reduction or elimination of Campylobacter in poultry production will have a significant impact on food safety and public health. Despite the significant progress made over the last decades, many puzzles remain about the epidemiology of Campylobacter on poultry farms, hampering the development of an effective control strategy. This longitudinal study was conducted to determine the prevalence and genetic diversity of Campylobacter in a U.S. commercial broiler production farm system. Cecal contents (15 samples/flock) and boot swabs (3 samples/flock) were collected from approximately 6-wk-old birds from 406 conventional broiler flocks reared in 53 houses on 15 farms (located within a relatively close geographic proximity and managed by the same poultry integrator) for up to eight consecutive production cycles and cultured for Campylobacter. Pulsed-field gel electrophoresis was used to investigate the genetic diversity of the Campylobacter jejuni isolates recovered from the cecal contents. The prevalence of Campylobacter at the farm, house, and flock levels were found to be 93% (14/15), 79% (42/53), and 47% (192/406), respectively. Campylobacter prevalence varied remarkably among different farms and flocks, with some farms or houses testing consistently negative while others being positive all the time over the entire study period. Campylobacter isolation rate changed significantly by sample type (higher by cecal contents vs. boot swabs) and season/production cycle (higher in spring vs. other seasons). The majority (88%; 2364/2675) of the isolates were identified as C. jejuni, and almost all the rest (11%; 303/2675) were Campylobacter coli. Genotyping showed limited diversity within a flock and suggested persistence of some C. jejuni clones over multiple production cycles on the same farm. In conclusion, this study indicated that although Campylobacter prevalence was overall high, there were marked differences in the prevalence among the broiler flocks or farms tested. Future studies aimed at identification of potential risk factors associated with differential Campylobacter status are warranted in order to develop effective on-farm interventions.

Influence and Role of the Body Mass Index and Its Development on the Course of Pediatric HSCT - a Retrospective Analysis

T Stetter and M Döring contributed equally to this work. Background Children and adolescents are at increased risk of malnutrition during the course of hematopoietic stem cell transplantation (HSCT). Increased endocrine activity, a catabolic metabolic state and inflammatory processes increase the energy metabolism and therapy-associated side effects impair nutritional intake. Studies have shown that the nutritional status before HSCT in adults has a decisive influence on the time afterwards. The role of nutritional status in children and adolescents before, during, and after HSCT remains poorly understood. Patients and Methods In this monocentric, retrospective study, data from patients who underwent autologous or allogeneic HSCT at the University Children's Hospital Tübingen between November 2012 and November 2021, were analyzed. Exclusion criteria were known gastrointestinal malformations, permanent enteral/parenteral nutrition, and known chronic inflammatory bowel disease before HSCT. On three observation days (baseline: day of in-patient admission, day +30, and day +200 after HSCT) data on age-adjusted BMI percentiles, incidence of transplant-associated adverse events (TRAE; e.g., graft-versus-host disease (GvHD), veno-occlusive disease (VOD), mucositis, infection), and survival (up to 10 years after HSCT; last note in file). were collected. Patients were categorized into alloHSCT and autoHSCT Patients as well as BMI-percentile groups depending on baseline BMI percentile: &amp;lt;3rd percentile, 3rd-&amp;lt;10th percentile, 10th-&amp;lt;25th percentile, 25th-&amp;lt;50th percentile, 50th-&amp;lt;75th percentile, 75th-97th percentile, and &amp;gt;97th percentile. Results A total of 365 patients with a median age of 9 years (0-23 years), 56% of whom were female, were studied. Of these, 307 (84%) underwent allogeneic and 58 (16%) autologous HSCT. The underlying disease was malignant in 262 cases (72%) and non-malignant in 103 cases (28%). The proportions of patients fell into the corresponding BMI percentile groups at the respective observation days (see Table). During the course of time until day +200 after HSCT, the BMI percentile category remained the same in 28% of the cases, in 17%, 10%, and 3% of cases, respectively, patients moved up one, two, and &amp;gt;2 categories, respectively; in 20%, 12%, and 9% of cases, patients dropped one, two, and &amp;gt;2 categories, respectively, compared to baseline. However, comparing the alloHSCT and autoHSCT patients, a different distribution in the development until day +200 is noticeable. Overall, 27% of the alloHSCT and 44% of autoHSCT patients ascend one or more percentile groups, whereas 46% of the alloHSCT and 22% of the autoHSCT patients descend one or more BMI percentile categories. Higher-grade intestinal GvHD stage 3-4 occurred statistically more frequently in the percentile group &amp;gt;75th-97th ( p=0.0028), but when all grades were considered together, no significant clustering in any group was observed. A significant accumulation ( p=0.0413) of VOD, BKV cystitis ( p=0.0021), and Viremia ( p=0.0081) was found in the group &amp;gt;75th-97th percentile. A total of 115 patients (32%) died during the observation period. In the alloHSCT group, 35 deaths (12% of alloHSCT patients) fell under TRM. Here, of the 35 deceased, 12 (34% of deceased, 4% of total alloHSCT patients) were ascribed to the &amp;gt;75th-97th percentile group; the median proportion of deceased patients was 9% (1% of alloHSCT group) in the other percentile groups. In the autoHSCT group, a total of 23 patients (41%) died; of these, all were due to progression or recurrence of the underlying disease. The &amp;lt;3rd percentile and &amp;gt;25th-50th percentile groups each recorded the most deaths, with 26% of those who died and 11% of patients in the autoHSCT group, respectively. Conclusions Contrary to the expectation that particularly underweight pediatric HSCT patients might be more affected by complications, this data collection showed statistically clustered complications and TRM in the group of patients between the 75th-97th BMI percentiles. Although this is only a status description of body weight and length during follow-up, and not a specific survey of nutritional status, the data analysis suggests that HSCT patients in the overweight range should be focused on more closely. A prospective survey of differential nutritional status in pediatric HSCT patients is currently being conducted at our study center.

Mapping Chemo-Resistance Profiles of Pediatric Acute Leukemia through Integration of Ex-Vivo Drug Screens with Molecular Data

Introduction While novel approaches for treatment of acute lymphoblastic leukemia (ALL) in children have resulted in significant improvements in survival rates, survivors are faced with potentially life-long challenges as a result of the highly toxic treatment and the outcome for patients who relapse remains poor. The current risk-adapted treatment for ALL in children includes multiple drugs administered sequentially or in combination, with little variation in timing or intensity. Factors that affect varying treatment response between patients and cellular mechanisms that underly response to individual drugs are still under exploration. In this study, we investigate the relationship between ex-vivo drug resistance, transcriptomes, epigenomes and clinical outcome, with the aim of better understanding underlying determinants of treatment response. Methods Pediatric patients diagnosed with B-ALL between 1992 and 2008 in the Nordic countries were included in the study (n = 598). For these patients, ex-vivo drug response to ten clinically used drugs was determined using the fluorometric microculture cytotoxicity assay (FMCA). Clinical data, including follow-up information (overall survival and relapse free survival) was available for all patients. Additionally, DNA-methylation (450K array, n = 383) and gene expression profiling (RNA-seq, n = 119) was performed on a subset of diagnostic patient samples. The patients were stratified into low, medium and high resistance groups based on ex-vivo drug response. The low and high resistance groups were compared with regard to differentially methylated CpG sites or expressed genes associated with individual drug response profiles. Results Overall survival was significantly lower for patients with higher ex-vivo drug resistance to dexamethasone, doxorubicin and thioguanine compared to patients that displayed sensitivity to these drugs (p &amp;lt; 0.05). Similarly, relapse free survival was significantly lower for patients with ex-vivo resistance to cytarabine, dexamethasone, doxorubicin, prednisolone and thioguanine (p &amp;lt; 0.05). After adjusting for sex and risk group, according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocol, in a Cox proportional hazards model, higher hazard ratios for relapse were observed in the highly resistant group across all drugs. We identified distinct DNA methylation and gene expression patterns in patients with low and high resistance, indicating the presence of predetermined molecular resistance fates. These resistance patterns included 665 genes that were differentially expressed (absolute log2FC &amp;gt; 1, Benjamini-Hochberg adjusted p value &amp;lt; 0.05) and 1 422 CpG sites with differential methylation status (absolute mean β-value difference &amp;gt; 0.2, Benjamini-Hochberg adjusted p value &amp;lt; 0.05) in resistant compared to sensitive groups, for one or multiple drugs. With the aim of identifying the genes with the largest impact on drug resistance, we selected the most differentially expressed genes and the genes with the largest difference in methylation status between resistant and sensitive samples. Among these, we identified ~20 putative genes of interest for future studies into resistance mechanisms based on potential functional prioritization. Conclusion Our findings confirm that drug response profiles are associated with outcome in pediatric ALL, reflecting response in vivo. We show molecular evidence for differentiating low- and high-resistant types based on diagnostic gene expression and DNA methylation profiles, suggesting contribution of certain molecular signatures to drug response. Our data integrating molecular data with drug response profiles may help identifying such changes of importance in drug resistance mechanisms.

Assessment of plasma melatonin profile in summer anestrous buffaloes exhibiting differential fertility following melatonin implants treatment

Plasma melatonin vis-a-vis anestrus or estrus status of buffaloes during summer or winter season (n=50 in each group) suggested its impact on reproductive axis as plasma melatonin activity tended to be higher (P&gt;0.05) in buffaloes exhibiting estrus compared to their anestrous counterparts irrespective of season. The differential reproductive status of these buffaloes was confirmed by history as well as differences (P&lt;0.05) in diameter of largest follicle and luteal profile. Further, for evaluating the impact of melatonin treatment during summer season on reproductive axis, 132 anestrous buffaloes were subcutaneously inserted 2x4 mm absorbable slow-release melatonin implants (18 mg/50 kg b wt) at the base of left ear and 60 buffaloes were used as control. In these buffaloes, ovarian ultrasonography and jugular vein blood sampling was carried out at 7-day interval till day 35 post-treatment or till ovulation, whichever was earlier. Control and implanted buffaloes were subjected to artificial insemination (AI) at overt or induced estrus followed by pregnancy diagnosis at day 90 post-AI. In treatment group, an increase (P&lt;0.05) in plasma melatonin was recorded in all the buffaloes, exhibiting differential fertility status, during post-treatment study period compared to their pre-treatment and control group values. However, within treatment group, there was no difference (P&gt;0.05) in plasma melatonin between ovulatory or non-ovulatory as well as between pregnant or non-pregnant counterparts. Moreover, plasma melatonin within control buffaloes remained similar (P&gt;0.05) throughout the study period irrespective of differential exhibition of fertility. It can be concluded that factors other than circulating melatonin are also involved in the display of differential fertility in terms of initiation of ovarian cyclicity/ovulation or ability to conceive in summer anestrous buffaloes.

Exotic herbivores and fire energy drive standing herbaceous biomass but do not alter compositional patterns in a semiarid savanna ecosystem

AbstractQuestionsFire regime alterations are pushing open ecosystems worldwide past tipping points where alternative steady states characterized by woody dominance prevail. This reduces the frequency and intensity of surface fires, further limiting their effectiveness for controlling cover of woody plants. In addition, grazing pressure (exotic or native grazers) can reinforce woody encroachment by potentially reducing fine‐fuel loads. We investigated the effects of different fire energies on the herbaceous plant community, together with mammalian wildlife herbivory (exotic and native combined) exclusion, to inform best management practices.LocationTexas semi‐arid savanna, southern Great Plains, USA.MethodsWe conducted an experiment in which we manipulated fire intensity and herbivore access to herbaceous biomass in a split‐plot design. We altered fire energy via fuel addition rather than applying fire under different environmental conditions to control for differences in standing biomass and composition attributable to differential plant physiological status and fire season.ResultsHigh‐energy fire did not reduce herbaceous biomass or alter plant community composition, although it did increase among‐plot variability in composition and forb biomass relative to low‐energy fire and non‐burned controls. Grazing pressure from native and non‐native mammalian herbivores reduced above‐ground herbaceous biomass regardless of fire treatments, but did not alter community composition.ConclusionsManagers seeking to apply high‐intensity prescribed fire to reduce woody encroachment will not negatively impact herbaceous plant productivity or alter community composition. However, they should be cognizant that repeated fires necessary for greatly reducing woody plants in heavily invaded areas might be difficult to accomplish due to fine‐fuel reduction from wild herbivores. High fencing to restrict access by wildlife herbivores or culling might be necessary to build fuels sufficient to conduct high‐intensity burns for woody‐plant reduction.

From Developing Country Leader to Flexible Negotiator: New Directions in Brazilian Trade Strategy

AbstractBrazil has changed its negotiation strategy in World Trade Organization (WTO) negotiations. In the first half of the WTO era (1995–), Brazil adopted a strong developing country leadership role as coordinator and spokesperson of the G20 group of developing countries. More recently, however, this group has disappeared from the negotiation scene. This article examines how Brazil has departed from a 2000 status quo and arrived at a more flexible approach, less reliant on the industrialized-developing divide as a structuring principle of its diplomacy. Using WTO negotiation documents, trade delegate interviews, dispute settlement case law, and secondary literature, I outline the contours of new directions in Brazilian trade policy. These include joint legislative initiatives with the EU, a move towards the plurilateral level on non-traditional issues, a greater heterogeneity of dispute settlement targets, and a newly flexible handling of its rights under the WTO's special and differential treatment status. The article contributes to ongoing debates on Brazil's status in international affairs, its reliance on large coalitions, and the maintenance of followership as key directives of its foreign policy, and scholarship that sees Brazil as stuck in a ‘graduation dilemma’.