UNASSIGNED: Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor of the biliary tract that is prone to recurrence and metastasis and is characterized by poor sensitivity to chemotherapy and overall prognosis. For these reasons, there is an urgent need to understand its pathological mechanisms and develop effective treatments. To address this challenge, the establishment of suitable preclinical models is critical.
UNASSIGNED: Fresh ICC tissue samples were used for primary culture and subculture. The cell line was evaluated by cell proliferation assays, clonal formation assays, karyotype analysis, and short tandem repeat (STR) analysis. Drug resistances against oxaliplatin, paclitaxel, gemcitabine and 5-fluorouracil (5-FU) were evaluated by CCK-8 assay. Subcutaneous injection of 1 × 106 cells to three BALB/c nude mice was conducted for xenograft studies. The hematoxylin and eosin (H&E) staining was used to detect the pathological status of the cell line. The expression of biomarkers CK7, CK19, Ki-67, E-cadherin and vimentin was determined by immunocytochemistry assay.
UNASSIGNED: A new ICC cell line named ICC-X2 was successfully established. Like ICC-X3 established using the same patient\'s metastatic tumor, the cell line has been continuously cultured in vitro for more than a year and has been passaged more than 100 times. ICC-X2 retained the typical biliary epithelial morphology. The population doubling time of ICC-X2 is 48 h. The cells demonstrated an abnormal nearly tetraploid karyotype. The STR analysis confirmed that ICC-X2 was highly consistent with the primary tumor tissue and not cross-contaminated by existing cell lines. ICC-X2 cells positively expressed CK7, CK19, E-cadherin, and vimentin, and the positive expression of Ki-67 in ICC-X2 cells was 40%. The ICC-X2 cells exhibited a strong clonogenic ability. The drug sensitivity test indicated that ICC-X2 was sensitive to oxaliplatin and paclitaxel, but naturally resistant to gemcitabine and 5-FU. ICC-X2 was rapidly able to form transplanted tumors in vivo after subcutaneous inoculation in nude mice.
UNASSIGNED: ICC-X2 is an excellent experimental model that can be used for studying the occurrence, development, and metastasis of ICC and investigating the mechanism of tumor drug resistance.

BACKGROUND: Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice. These cells consist of both epithelial and mesenchymal cells. Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma. However, cholangiocarcinoma sarcoma cell lines are not available in cell banks.
OBJECTIVE: To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line, namely CBC2T-2.
METHODS: We conducted a short tandem repeat (STR) test to confirm the identity of the CBC2T-2 cell line. Furthermore, we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies. The tumorigenic potential of CBC2T-2 cells was tested in vivo using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The cells were injected subcutaneously and tumor formation was observed. In addition, immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts. The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma. Lastly, whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line.
RESULTS: The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue. The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology. The cells exhibited a high proliferation ratio with a doubling time of 47.11 h. This cell line has migratory, invasive, and clonogenic abilities. The chromosomes in the CBC2T-2 cells were polyploidy, with numbers ranging from 69 to 79. The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice. CBC2T-2 cells and xenografts were positive for both the epithelial marker, CK19, and the mesenchymal marker, vimentin. These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics. The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma, and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option.
CONCLUSIONS: We established the first human cholangiocarcinoma sarcoma cell line, CBC2T-2, with stable biogenetic traits. This cell line, as a research model, has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.

Gastrointestinal nematodes (GIN) use flexible life history strategies to maintain their fitness under environmental challenges. Costs incurred by a challenge to one life trait can be recouped by increasing the expression of subsequent life traits throughout their life cycle. Anticipating how parasites respond to the challenge of control interventions is critical for the long-term sustainability of the practice and to further ensure that the parasites withstand favourable adaptive responses. There is currently limited information on whether distinct populations of a GIN species respond to the same environmental challenge in a consistent manner, with similar alterations to their life history strategies or comparable fitness outcomes. This study compared the life history traits and experimental fitness of three distinct Haemonchus contortus isolates exposed to environmental challenges at both the parasitic (i.e., passage through resistant or susceptible sheep) and free-living (i.e., exposure to diverse climatic conditions) life stages. The key findings show that H. contortus maintain their fitness under challenge with isolate-specific alterations to their life history strategies. Further, partial exploration of the H. contortus isolates transcriptomes using cDNA-AFLP methods confirmed disparate expression profiles between them. These results bring fresh insights into our understanding of the non-genetic adaptive processes of GIN that may hinder the efficacy of parasite control strategies.

Drug resistance is an urgent problem to be solved in the treatment of non‑small‑cell lung cancer (NSCLC). Osimertinib is a third‑generation EGFR‑tyrosine kinase inhibitor, which can improve the efficacy and quality of life of patients; however, the inevitable resistance after long‑term use of osimertinib often leads to treatment failure. Cell lines are key tools for basic and preclinical studies. At present, few osimertinib‑resistant cell lines (HCC827‑OR and H1975‑OR) have been established. In the present study, osimertinib‑resistant cell lines were established by gradually increasing the drug concentration. Half‑maximal inhibitory concentration (IC50), cell morphology, whole exon sequencing, Cell Counting Kit‑8 assay, EdU staining and flow cytometry were used to evaluate the osimertinib‑resistant cell lines. Western blot analysis was used to detect the expression levels of key proteins involved in osimertinib resistance. The circular RNA (circRNA) expression profile was identified by RNA sequencing (RNA‑seq) analysis of HCC827, HCC827‑OR, H1975 and H1975‑OR cells. Subsequently, the biological roles of differentially expressed circRNAs were explored in in vitro studies. Osimertinib‑resistant cell lines were successfully established via treatment with an increasing concentration of osimertinib. Osimertinib IC50 and proliferation of resistant cells were much higher than those of sensitive cells. Notably, phosphorylated (p)‑AKT and p‑ERK were markedly activated in resistant cells, and the inhibitory effect of osimertinib on p‑AKT and p‑ERK was weaker in resistant cells than that in parental cells. RNA‑seq analysis identified differentially expressed circRNAs in HCC827, HCC827‑OR, H1975 and H1975‑OR cells. The most dysregulated circRNAs (circPDLIM5 and circPPP4R1) were selected for further functional study. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the host genes of differentially expressed circRNAs were associated with \'endocytosis\' and \'regulation of autophagy\'. In conclusion, the present study established osimertinib‑resistant cell lines and revealed that circRNAs may serve as a promising biomarker in NSCLC osimertinib resistance.

The timing of the establishment of the HIV latent viral reservoir (LVR) is of particular interest, as there is evidence that proviruses are preferentially archived at the time of antiretroviral therapy (ART) initiation. Quantitative viral outgrowth assays (QVOAs) were performed using Peripheral Blood Mononuclear Cells (PBMC) collected from Ugandans living with HIV who were virally suppressed on ART for >1 year, had known seroconversion windows, and at least two archived ART-naïve plasma samples. QVOA outgrowth populations and pre-ART plasma samples were deep sequenced for the pol and gp41 genes. The bayroot program was used to estimate the date that each outgrowth virus was incorporated into the reservoir. Bayroot was also applied to previously published data from a South African cohort. In the Ugandan cohort (n = 11), 87.9 per cent pre-ART and 56.3 per cent viral outgrowth sequences were unique. Integration dates were estimated to be relatively evenly distributed throughout viremia in 9/11 participants. In contrast, sequences from the South African cohort (n = 9) were more commonly estimated to have entered the LVR close to ART initiation, as previously reported. Timing of LVR establishment is variable between populations and potentially viral subtypes, which could limit the effectiveness of interventions that target the LVR only at ART initiation.

As a key partner of Ministry of Health (MOH) Ethiopia, The Clinton Health Access Initiative (CHAI) had been implementing the Child Survival Project (CSP) since October 2015. Strengthening DTC was one of its focuses to improve overall supply chain management (SCM). The objective of this study are to review the evolution of DTCs in Ethiopia from their early years to current practice and identify the major driving and hindering factors for their functionality. A descriptive mixed study design was employed. The study made use of qualitative data supplemented with quantitative data, generated from both primary and secondary sources through key informant interviews and desk review methods. DTCs were introduced in Ethiopia in the early 1980s. The mandate of DTCs has been given to four different government organizations during that time. As a result, due to a lack of coordination among these organizations, its implementation was lagging. Recently, the government and its partners have given attention to DTCs. More than 5847 professionals underwent DTC training from 2016 onwards. DTC establishment in health facilities improved from 85% to 98% between 2015 and 2019 during baseline and end-line assessments carried out by CHAI/CSP. Similarly, DTC functionality in HFs improved from 20% to 63%. The CHAI/CSP regular supervision data analysis revealed that DTC establishment improved from 83% to 100% of HFs, while its functionality improved from 5% to 72% between 2016 and 2019, respectively. A chi-square test of independence examining the relationship between facility and pharmacy head training on DTCs and functionality of DTC in the same facility revealed a significant association between the two variables at p<0.0001. Conclusions: Providing consistent capacity building and availing strong monitoring and evaluation system improves functionality of DTCs. Moreover, national coordinating bodies for DTCs and similar structures at Regional Health Bureaus and woreda health offices should be established.

Biomass crops provide significant potential to substitute for fossil fuels and mitigate against climate change. It is widely acknowledged that significant scale up of biomass crops is required to help reach net zero targets. Miscanthus is a leading biomass crop embodying many characteristics that make it a highly sustainable source of biomass but planted area remains low. Miscanthus is commonly propagated via rhizome, but efficient alternatives may increase uptake and help diversify the cultivated crop. Using seed-propagate plug plants of Miscanthus has several potential benefits such as improving propagation rates and scale up of plantations. Plugs also provide an opportunity to vary the time and conditions under protected growth, to achieve optimal plantlets before planting. We varied combinations of glasshouse growth period and field planting dates under UK temperate conditions, which demonstrated the special importance of planting date on yield, stem number and establishment rates of Miscanthus. We also propagated Miscanthus in four different commercial plug designs that contained different volumes of substrate, the resulting seedlings were planted at three different dates into field trials. In the glasshouse, plug design had significant effects on above and belowground biomass accumulation and at a later time point belowground growth was restricted in some plug designs. After subsequent growth in the field, plug design and planting date had a significant effect on yield. The effects of plug design on yield were no longer significant after a second growth season but planting date continued to have a significant effect. After the second growth year, it was found that planting date had a significant effect on surviving plants, with the mid-season planting producing higher survival rates over all plug types.Establishment was positively correlated with DM biomass produced in the first growth season. Sowing date had a significant effect on establishment but the impacts of plug design were more nuanced and were significant at later planting dates. We discuss the potential to use the flexibility afforded by seed propagation of plug plants to deliver significant impacts in achieving high yield and establishment of biomass crops during the critical first two years of growth.

In this work, we use Drosophila melanogaster as a model to identify bacterial genes necessary for bacteria to colonize their hosts independent of the bulk flow of diet. Early work on this model system established that dietary replenishment drives the composition of the D. melanogaster gut microbiota, and subsequent research has shown that some bacterial strains can stably colonize, or persist within, the fly independent of dietary replenishment. Here, we reveal transposon insertions in specific bacterial genes that influence the bacterial colonization persistence phenotype by using a gene association approach. We initially established that different bacterial strains persist at various levels, independent of dietary replenishment. We then repeated the analysis with an expanded panel of bacterial strains and performed a metagenome-wide association (MGWA) study to identify distinct bacterial genes that are significantly correlated with the level of colonization by persistent bacterial strains. Based on the MGWA study, we tested if 44 bacterial transposon insertion mutants from 6 gene categories affect bacterial persistence within the flies. We identified that transposon insertions in four flagellar genes, one urea carboxylase gene, one phosphatidylinositol gene, one bacterial secretion gene, and one antimicrobial peptide (AMP) resistance gene each significantly influenced the colonization of D. melanogaster by an Acetobacter fabarum strain. Follow-up experiments revealed that each flagellar mutant was nonmotile, even though the wild-type strain was motile. Taken together, these results reveal that transposon insertions in specific bacterial genes, including motility genes, are necessary for at least one member of the fly microbiota to persistently colonize the fly. IMPORTANCE Despite the growing body of research on the microbiota, the mechanisms by which the microbiota colonizes a host can still be further elucidated. This study identifies bacterial genes that are associated with the colonization persistence phenotype of the microbiota in Drosophila melanogaster, which reveals specific bacterial factors that influence the establishment of the microbiota within its host. The identification of specific genes that affect persistence can help inform how the microbiota colonizes a host. Furthermore, a deeper understanding of the genetic mechanisms of the establishment of the microbiota could aid in the further development of the Drosophila microbiota as a model for microbiome research.

BACKGROUND: Although urological specialist practices are central pillars of outpatient care, there is a lack of current data on the care structure of these practices. A description of the structures in large cities versus rural areas as well as gender effects and generational differences is needed not only as a baseline measure for further studies.
METHODS: The survey includes data from the physician directory of the Stiftung Gesundheit as well as from the German Medical Association and the Federal Statistical Office. Colleagues were divided into subgroups. Based on the different subgroup sizes, statements about the care structure of outpatient urology in Germany can be made.
RESULTS: While the majority of urologists in larger cities work in professional practice groups and care for fewer patients on average, in rural areas there is a particularly high proportion of individual practices with more inhabitants to be cared for per urologist. Female urologists work more frequently in the context of inpatient care. When female urology specialists choose to establish themselves, they are more likely to do so in practice groups and in urban areas. In addition, there is a shift in gender distribution: the younger the age subgroup considered, the higher the proportion of female urologists among all colleagues.
CONCLUSIONS: This study is the first to describe the current structure of outpatient urology care in Germany. Future trends are already emerging that will significantly influence our way of working and the care of patients in the coming years.
UNASSIGNED: HINTERGRUND: Obwohl urologische Facharztpraxen zentraler Pfeiler der ambulanten Patientenversorgung in Deutschland sind, fehlen hierzu aktuelle Daten. Eine Beschreibung der Strukturen in Großstädten gegenüber dem ländlichen Raum sowie von Gendereffekten und Generationsunterschieden ist auch als Ausgangsbasis für weitere Untersuchungen erforderlich.
METHODS: Die Erhebung umfasst Daten des Arztverzeichnisses der Stiftung Gesundheit sowie der Bundesärztekammer und des Statistischen Bundesamtes. Die erfassten Praxen bzw. Kolleg*innen wurden in Subgruppen eingeteilt. Anhand der unterschiedlichen Subgruppengröße lassen sich Aussagen über die Versorgungsstruktur der ambulanten Urologie in Deutschland treffen.
UNASSIGNED: Während in größeren Städten die Mehrheit der niedergelassenen Urolog*innen in Berufsausübungsgemeinschaften (BAG) arbeitet und durchschnittlich weniger Patient*innen betreut, zeigt sich im ländlichen Raum ein besonders hoher Anteil an Einzelpraxen mit mehr zu versorgenden Einwohnern pro Urolog*in. Urologinnen arbeiten häufiger in der stationären Versorgung. Wenn sich Fachärztinnen für Urologie für die Niederlassung entscheiden, dann eher in BAG und eher in städtischen Gebieten. Außerdem zeigt sich eine Verschiebung der Geschlechterverteilung: Je jünger die betrachtete Alterssubgruppe, desto höher liegt der Anteil an Urologinnen unter allen Kolleg*innen.
UNASSIGNED: Wir beschreiben erstmalig die tatsächliche Versorgungsstruktur der ambulanten Urologie in Deutschland. Hierbei deuten sich Trends an, die die Art zu arbeiten und die Patientenversorgung in einigen Jahren maßgeblich beeinflussen werden.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest risk assessment of Amyelois transitella (Lepidoptera: Pyralidae), the navel orangeworm, for the EU. The quantitative assessment considered two scenarios: (i) current practices and (ii) a requirement for chilled transport. The assessment focused on pathways of introduction, climatic conditions and cultivation of hosts allowing establishment, spread and impact. A. transitella is a common pest of almonds, pistachios and walnuts in California, which is the main source for these nuts imported into the EU. Based on size of the trade and infestation at origin, importation of walnuts and almonds from the USA was identified as the most important pathways for entry of A. transitella. Using expert knowledge elicitation (EKE) and pathway modelling, a median estimate of 2,630 infested nuts is expected to enter the EU each year over the next 5 years (90% certainty range (CR) from 338 to 26,000 infested nuts per year). However, due to estimated small likelihoods of transfer to a host, mating upon transfer and survival of founder populations, the number of populations that establish was estimated to be 0.000698 year-1 (median, 90% CR: 0.0000126-0.0364 year-1). Accordingly, the expected period between founding events is 1,430 years (median, 90% CR: 27.5-79,400 year). The likelihood of entry resulting in establishment is therefore considered very small. However, this estimate has high uncertainty, mainly concerning the processes of transfer of the insect to hosts and the establishment of founder populations by those that successfully transfer. Climate matching and CLIMEX modelling indicate that conditions are most suitable for establishment in the southern EU, especially around the Mediterranean basin. The median rate of natural spread was estimated to be 5.6 km/year (median, 90% CR 0.8-19.3 km/year), after an initial lag period of 3.1 year (mean, 90% CR 1.7-6.2 year) following the establishment of a founder population. If A. transitella did establish, estimated median yield losses in nuts were estimated to be in the order of 1-2% depending on the nut species and production system. A scenario requiring imports of nuts to be transported under chilled conditions was shown to provide potential to further reduce the likelihood of entry.