Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factors phenol-soluble-modulins (PSMs) produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains induce tolerogenic DCs upon Toll-like receptor activation via the p38-CREB-IL-10 pathway in vitro. Here, we addressed the hypothesis that S. aureus PSMs disturb the adaptive immune response via modulation of DC subsets in vivo. Using a systemic mouse infection model we found that S. aureus reduced the numbers of splenic DC subsets, mainly CD4+ and CD8+ DCs independently of PSM secretion. S. aureus infection induced upregulation of the C-C motif chemokine receptor 7 (CCR7) on the surface of all DC subsets, on CD4+ DCs in a PSM-dependent manner, together with increased expression of MHCII, CD86, CD80, CD40, and the co-inhibitory molecule PD-L2, with only minor effects of PSMs. Moreover, PSMs increased IL-10 production in the spleen and impaired TNF production by CD4+ DCs. Besides, S. aureus PSMs reduced the number of CD4+ T cells in the spleen, whereas CD4+CD25+Foxp3+ regulatory T cells (Tregs) were increased. In contrast, Th1 and Th17 priming and IFN-γ production by CD8+ T cells were impaired by S. aureus PSMs. Thus, PSMs from highly virulent S. aureus strains modulate the adaptive immune response in the direction of tolerance by affecting DC functions.

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as "eat me" signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as "Trojan horse" vehicles for the efficient dispersion and replication of the bacterium within the host.

Wheat genetic improvement by integration of advanced genomic technologies is one way of improving productivity. To facilitate the breeding of economically important traits in wheat, SNP loci and underlying candidate genes associated with the 36 agro-morphological traits were studied in a diverse panel of 404 genotypes. By using Breeders' 35K Axiom array in a comprehensive genome-wide association study covering 4364.79 cM of the wheat genome and applying a compressed mixed linear model, a total of 146 SNPs (-log10 P ≥ 4) were found associated with 23 traits out of 36 traits studied explaining 3.7-47.0% of phenotypic variance. To reveal this a subset of 260 genotypes was characterized phenotypically for six quantitative traits [days to heading (DTH), days to maturity (DTM), plant height (PH), spike length (SL), awn length (Awn_L), and leaf length (Leaf_L)] under five environments. Gene annotations mined ∼38 putative candidate genes which were confirmed using tissue and stage specific gene expression data from RNA Seq. We observed strong co-localized loci for four traits (glume pubescence, SL, PH, and awn color) on chromosome 1B (24.64 cM) annotated five putative candidate genes. This study led to the discovery of hitherto unreported loci for some less explored traits (such as leaf sheath wax, awn attitude, and glume pubescence) besides the refined chromosomal regions of known loci associated with the traits. This study provides valuable information of the genetic loci and their potential genes underlying the traits such as awn characters which are being considered as important contributors toward yield enhancement.

Stomatal conductance (g s) in terrestrial vegetation regulates the uptake of atmospheric carbon dioxide for photosynthesis and water loss through transpiration, closely linking the biosphere and atmosphere and influencing climate. Yet, the range and pattern of g s in plants from natural ecosystems across broad geographic, climatic, and taxonomic ranges remains poorly quantified. Furthermore, attempts to characterize g s on such scales have predominantly relied upon meta-analyses compiling data from many different studies. This approach may be inherently problematic as it combines data collected using unstandardized protocols, sometimes over decadal time spans, and from different habitat groups. Using a standardized protocol, we measured leaf-level g s using porometry in 218 C3 woody angiosperm species in natural ecosystems representing seven bioclimatic zones. The resulting dataset of 4273 g s measurements, which we call STraits (Stomatal Traits), was used to determine patterns in maximum g s (g smax) across bioclimatic zones and whether there was similarity in the mean g smax of C3 woody angiosperms across ecosystem types. We also tested for differential g smax in two broadly defined habitat groups - open-canopy and understory-subcanopy - within and across bioclimatic zones. We found strong convergence in mean g smax of C3 woody angiosperms in the understory-subcanopy habitats across six bioclimatic zones, but not in open-canopy habitats. Mean g smax in open-canopy habitats (266 ± 100 mmol m-2 s-1) was significantly higher than in understory-subcanopy habitats (233 ± 86 mmol m-2 s-1). There was also a central tendency in the overall dataset to operate toward a g smax of ∼250 mmol m-2 s-1. We suggest that the observed convergence in mean g smax of C3 woody angiosperms in the understory-subcanopy is due to a buffering of g smax against macroclimate effects which will lead to differential response of C3 woody angiosperm vegetation in these two habitats to future global change. Therefore, it will be important for future studies of g smax to categorize vegetation according to habitat group.

Cassava brown streak disease (CBSD) is a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. This necrosis can encompass large areas of the root, rendering it inedible so that the entire cassava harvest can be lost. African cassava varieties are susceptible to either of the two viruses causing the disease, cassava brown streak virus (CBSV) and Uganda cassava brown streak virus, and while there are less sensitive varieties, all cassava eventually succumb to the disease. The lack of CBSD resistance in African cassava varieties prompted this search for new sources of virus resistance in the diversity of South American cassava germplasm held in the collection at International Center for Tropical Agriculture, Columbia. Our search for CBSD resistance in South American cassava germplasm accessions revealed that most of the 238 South American cassava lines infected with CBSV established systemic virus infections with moderate to severe disease symptoms on leaves and stems. Fifteen cassava accessions did not become virus infected, remained free of symptoms, and CBSV was undetected by qRT-PCR. When tuberous roots of those lines were examined, necrotic tissue was found in eight lines and CBSV was detected. The remaining seven cassava accessions remained clear of symptoms on all tissues and organs and were virus free. A broad spectrum of virus resistance also including other virus isolates was confirmed for the breeding lines DSC167 and DSC118. While detailed infection experiments with other cassava lines selected for resistance are still ongoing, this indicates that the resistance identified may also hold against a broader diversity of CBSVs. Taken together, we present the results of a comprehensive study on CBSV resistance and susceptibility in cassava germplasm accessions from South America. The virus resistance in cassava germplasm identified provides compelling evidence for the invaluable contribution of germplasm collections to supply the genetic resources for the improvement of our crops.

Autophagy (self-eating), a conserved pathway in eukaryotes, which is designed to handle cytoplasmic material in bulk and plays an important role in the remobilization of nutrient, such as nitrogen (N) under suboptimal nutrient conditions. Here, we identified a core component of an autophagy gene in rice (Oryza sativa), OsATG8a, with increased expression levels under N starvation conditions. Overexpression of OsATG8a significantly enhanced the level of autophagy and the number of effective tillers in the transgenic rice. In addition, the transgenic lines accumulated more N in grains than in the dry remains and the yield was significantly increased under normal N conditions. Further N allocation studies revealed that the nitrogen uptake efficiency (NUpE) and nitrogen use efficiency (NUE) significantly increased. Otherwise, under suboptimal N conditions, overexpression of OsATG8a did not seem to have any effect on yield and NUE, but NUpE was still improved significantly. Based on our findings, we consider OsATG8a to be a great candidate gene to increase NUE and yield.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg -/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg -/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft.

Given that most bladder cancers (BCs) are diagnosed in advanced stages with poor prognosis, this study aims to find novel biomarkers associated with the progression and prognosis in patients with BC. 1,779 differentially expressed genes (DEGs) between BC samples and normal bladder tissues were identified in total. Then, 24 DEGs were regarded as candidate hub genes by constructing a protein-protein interaction (PPI) network and a random forest model. Among them, six genes (BUB1B, CCNB1, CDK1, ISG15, KIF15, and RAD54L) were eventually identified by using five analysis methods (one-way Analysis of Variance analysis, spearman correlation analysis, distance correlation analysis, receiver operating characteristic curve, and expression values comparison), which were correlated with the progression and prognosis of BC. Moreover, the validation of hub genes was conducted based on GSE13507, Oncomine, and CBioPortal. Results of univariate Cox regression analysis showed that the expression levels of all the hub genes were influence features of overall survival (OS) and cancer specific survival (CSS) based on GSE13507, and we further established a six-gene signature based on the expression levels of the six genes and their Cox regression coefficients. This signature showed good potential for clinical application suggested by survival analysis (OS: Hazard Ratio = 0.484, 95%CI: 0.298-0.786; P = 0.0034; CSS: Hazard Ratio = 0.244, 95%CI: 0.121-0.493, P < 0.0001) and decision curve analysis. In conclusion, our study indicates that six hub genes have great predictive value for the prognosis and progression of BC and may contribute to the exploration of further basic and clinical research of BC.

Background: Brachytherapy (BT) is one of the techniques available for retreatment of patients with locally recurrent nasopharyng eal carcinoma (rNPC). In this study, we evaluated the treatment outcome and late toxicities of three-dimensional high-dose-rate brachytherapy (3D-HDR-BT) for patients with locally rNPC. Materials and Methods: This is a retrospective study involving 36 patients with histologically confirmed rNPC from 2004 to 2011. Of the 36 patients, 17 underwent combined-modality treatment (CMT) consisting of external beam radiotherapy (EBRT) followed by 3D-HDR-BT, while the other 19 underwent 3D-HDR-BT alone. The median dose of EBRT for the CMT group was 60 (range, 50-66) Gy, with an additional median dose of BT of 16 (range, 9-20) Gy. The median dose for the 3D-HDR-BT group was 32 (range, 20-36) Gy. The measured treatment outcomes were the 5- and 10-year locoregional recurrence-free survival (LRFS), disease-free survival (DFS), overall survival (OS), and late toxicities. Results: The median age at recurrence was 44.5 years. The median follow-up period was 70 (range, 6-142) months. The 5-year LRFS, DFS, and OS for the entire patient group were 75.4, 55.6, and 74.3%, respectively, while the 10-year LRFS, DFS, and OS for the entire patient group were 75.4, 44.2, and 53.7%, respectively. The 10-year LRFS in the CMT group was higher than that in the 3D-HDR-BT-alone group (93.8 vs. 58.8%, HR: 7.595, 95%CI: 1.233-61.826, p = 0.025). No grade 4 late radiotherapy-induced toxicities were observed. Conclusions: 3D-HDR-BT achieves favorable clinical outcomes with mild late toxicity in patients with locally rNPC.

Aim: Lung metastases from an extra-pulmonary origin occasionally present with a limited metastatic disease burden. In cases where metastatectomy is not feasible, stereotactic body radiation therapy (SBRT) represents a non-invasive, efficacious option. We report the outcomes of patients treated with lung SBRT in cases of limited metastatic disease. Methods: We retrospectively reviewed outcomes in 44 patients with 50 lung nodules from various extra-pulmonary malignancies treated with SBRT. Fifty percent of the patients were male and median age was 64. The median number of nodules was 1 and 90% of patients had oligometastatic disease. Thirty-four percent of patients had extra-thoracic disease. Results: Fifty lung nodules were treated with SBRT in 44 patients. Median dose was 48 Gy in 5 fractions with a median biological effective dose (BED) of 100 Gy10. Follow-up imaging was available for review in 96% of nodules. Median follow-up was 17.5 months. One year local control was 82%. BED >72 Gy10 predicted improved local control (90 vs. 57% at 1 year). One year overall survival following SBRT was 66%. There was no difference in overall survival if patients had extra-thoracic disease. Conclusion: Lung SBRT is a safe, effective tool for treatment of limited lung metastases. Dose selection remains important for local control.

The potential of neuroendocrine neoplasms (NEN) to respond to checkpoint inhibitors is largely unknown and full of great expectations. Immunohistochemical (IHC) studies of programmed cell death ligand 1 (PD-L1) expression in the tumor microenvironment and its implications in predicting the response to checkpoint inhibition is a very active subject. Currently, the combined analysis of PD-L1 expression and tumor-associated immune cell (TAIC) infiltration is considered the best predictive marker of therapeutic response. Here we investigated the expression of PD-L1 on tumor cells (TC) and tumor-infiltrating immune cells (IC) by IHC in 68 NEN samples with a high proliferation rate (Ki-67 >20%) from 57 patients and in 22 samples we correlated it with TAIC density by assessing intratumoral infiltration of CD3+, CD8+, and CD68+ cells. Furthermore, the tumor microenvironment was evaluated according to the classification of Teng et al. We detected PD-L1 expression in 31.6% of NEN G3. Its expression usually was weak and more IC than TC expressed PD-L1. The proportion of tumors positive for PD-L1 was comparable in NEN from different sites of origin but varied depending on tumor differentiation and disease extension. No positive IHC staining was found in 3 well-differentiated neuroendocrine tumors (NETs) with a proliferation rate above 20% (NET G3). When analyzing TAIC, we rarely (18.2%) detected intratumoral CD8+ cells, whereas infiltration by CD3+ and CD68+ cells was more common (45.5 and 59.1%, respectively). By combining CD3+ cells and PD-L1 status, we identified the immune ignorant phenotype of tumor microenvironment as being the most common phenotype, supporting the concept of a preferably combined immunotherapeutic approach in neuroendocrine carcinoma (NEC).

Background: Triple-negative breast cancer (TNBC) accounts for 12-20% of all breast cancers. Diagnosis of TNBC is sometimes quite difficult based on morphological assessment and immunohistochemistry alone, particularly in the metastatic setting with no prior history of breast cancer. Methods: Molecular profiling is a promising diagnostic approach that has the potential to provide an objective classification of metastatic tumors with unknown primary. In this study, performance of a novel 90-gene expression signature for determination of the site of tumor origin was evaluated in 115 TNBC samples. For each specimen, expression profiles of the 90 tumor-specific genes were analyzed, and similarity scores were obtained for each of the 21 tumor types on the test panel. Predicted tumor type was compared to the reference diagnosis to calculate accuracy. Furthermore, rank product analysis was performed to identify genes that were differentially expressed between TNBC and other tumor types. Results: Analysis of the 90-gene expression signature resulted in an overall 97.4% (112/115, 95% CI: 0.92-0.99) agreement with the reference diagnosis. Among all specimens, the signature correctly classified 97.6% of TNBC from the primary site (41/42) and lymph node metastasis (41/42) and 96.8% of distant metastatic tumors (30/31). Furthermore, a list of genes, including AZGP1, KRT19, and PIGR, was identified as differentially expressed between TNBC and other tumor types, suggesting their potential use as discriminatory markers. Conclusion: Our results demonstrate excellent performance of a 90-gene expression signature for identification of tumor origin in a cohort of both primary and metastatic TNBC samples. These findings show promise for use of this novel molecular assay to aid in differential diagnosis of TNBC, particularly in the metastatic setting.

Uterine corpus endometrial carcinomas (UCEC) are clinically divided into two subgroups-endometrioid endometrial carcinoma (EEC) or non-endometrioid endometrial carcinoma (NEEC). The first group shows relatively better prognosis. However, the discrimination seems to be insufficient due to the fact that in the mildest EEC are patients with poor treatment response and bad prognosis. Our aim was to examine the molecular background of such phenomenon and whether gene expression patterns might be of importance for the clinic. We focused our analysis on WNT pathway target genes since it is one of the main regulators of endometrial proliferation and differentiation. In silico analysis of TCGA data, including Weighted Co-expression Network Analysis, Principle Component Analysis, and Multiple Factor Analysis, allows to select 28 genes that serve as a predictive markers for UCEC patients. Our study revealed that there is a subgroup of the endometrioid cases that molecularly resembles mixed/serous groups. This may explain the reason for existence of subgroup of patients, that although clinically diagnosed with the mildest endometrioid UCEC type, yet present failure in treatment and aggressive course of the disease. Our study suggests that worse outcome in these patients may be based on a disruption of proper WNT signalling pathway resulting in deregulation of its effector genes. Moreover, we showed that mixed group consisting of tumours containing both endometrioid and serous types of cells, has serous expression profile of WNT targets. The proposed gene set allows to predict progression of the disease trough dividing patients into groups of low or high grade with 70.8% sensitivity and 88.6% specificity (AUC = 0.837) as well as could predict patient prognosis associated with UCEC subtype with 70.1% sensitivity and 86.2% specificity (AUC = 0.855). Relatively small number of implicated genes makes it highly applicable and possibly clinically simple and useful tool.

Vi capsular polysaccharide (Vi) is a major virulence factor of human typhoid-causing pathogen Salmonella enterica serovar Typhi (S. Typhi). It distinguishes S. Typhi from closely related non-typhoidal Salmonella serovars such as S. Typhimurium which do not normally cause systemic infection in humans. Vi not only forms a capsule around S. Typhi but it is also readily released from this pathogen. We have previously reported that Vi targets prohibitin to inhibit cellular responses activated through immune receptors. Here, we show that engagement of membrane prohibitin with Vi prevents Salmonella-induced activation of small Rho-family GTPases, Rac1, and Cdc42, and suppresses actin cytoskeletal rearrangements resulting in reduced invasion and highly subdued inflammatory responses. Cells infected with S. Typhimurium in the presence of Vi show poor activation of NF-kB and MAP-kinase pathways of intracellular signaling. Treatment with Vi brings about redistribution of Rac-1, prohibitin, and ganglioside GM1 in membrane raft domains. Vi-mediated interference with activation of Rho-family GTPases represents a previously unrecognized mechanism by which S. Typhi can limit its invasion and alarming of the host.

Introduction: It has been proposed that the use of cue-reminders may increase the effectiveness of interventions that aim to prevent health-risk behaviors (i.e., having unsafe sex, unhealthy dietary intake, lack of physical activity, and substance use). The aim of this systematic review was to explore whether there is evidence supporting this proposition, and to explore how cue-reminders are applied in health-risk behavior interventions to date. Method: We systemically reviewed (non-) randomized trials that examine differences in health-risk behaviors between an experimental group receiving an intervention with exposure to a cue-reminder and a control group receiving the intervention without such cue. Results: Six studies were eligible for inclusion. The studies differed in sample and research design, and how the cue-reminder was applied. One study demonstrated a positive and small effect, and one study found a negative medium effect of the cue-reminder. In the remaining studies, the effect sizes were positive but non-significant. Discussion: It is unclear whether complementing health-risk behavior interventions with cue-reminders increases the effectiveness of these interventions. Further investigation and experimentation into the efficiency and effectiveness of cue-reminders is needed before health-risk behavior interventions are complemented with cue-reminders.

Background: Polyethylene glycol (PEG) has been regarded as the primary recommendation for bowel preparation before colonoscopy. However, a conclusive conclusion has not yet been generated. Aim: We performed this updated meta-analysis to further investigate the comparative efficacy and safety of low volume preparation based on PEG plus ascorbic acid related to 4L PEG. Methods: A systematic search was conducted to retrieve potential randomized controlled trials (RCTs) in PubMed, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) from January 2000 to April 2018. Two independent searchers critically searched all potential citations, extracted data, and appraised risk of bias accordingly. Moreover, we used the STATA 12.0 and trial sequential analysis (TSA) 0.9 to complete all analyses. Results: A total of 13 RCTs enrolling 3,910 patients met inclusion criteria. Meta-analysis based on PP analysis indicated that compared to standard volume PEG regime, low volume regime improved patient compliance RR = 1.01; 95% CIs = 1.00, 1.03; P = 0.143 (≥75% intake); RR = 1.07; 95% CIs = 1.00, 1.14; P = 0.046 (100% intake), the willingness to repeat the same regime (RR = 1.30; 95% CIs = 1.07, 157; P = 0.007), and patient acceptability (RR = 1.18; 95% CIs = 1.07, 1.29; P = 0.001), and decreased the overall adverse events (RR = 0.86; 95% CIs = 0.77, 0.96; P = 0.009). However, no difference was observed between these two different solutions for bowel preparation efficacy (RR = 0.98; 95% CIs = 0.95, 1.02; P = 0.340). These all results were further confirmed by TSA. Conclusions: The effect of low volume regime was not inferior to the standard volume PEG regime, and low volume regime was associated with better compliance when subjects ingested all the solution, willingness to repeat the same regime, higher acceptability, and lower nausea in non-selected population.

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol-AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol-AmB structures at the cell surface, preventing AmB cytotoxicity.

Colorectal liver metastases (CRLM) exhibit distinct histopathological growth patterns (HGPs) that are indicative of prognosis following surgical treatment. This study aims to assess the reliability and replicability of this histological biomarker. Within and between metastasis HGP concordance was analysed in patients who underwent surgery for CRLM. An independent cohort was used for external validation. Within metastasis concordance was assessed in CRLM with ≥ 2 tissue blocks. Similarly, concordance amongst multiple metastases was determined in patients with ≥ 2 resected CRLM. Diagnostic accuracy [expressed in area under the curve (AUC)] was compared by number of blocks and number of metastases scored. Interobserver agreement (Cohen's k) compared to the gold standard was determined for a pathologist and a PhD candidate without experience in HGP assessment after one and two training sessions. Both the within (95%, n = 825) and the between metastasis (90%, n = 363) HGP concordance was high. These results could be replicated in the external validation cohort with a within and between metastasis concordance of 97% and 94%, respectively. Diagnostic accuracy improved when scoring 2 versus 1 blocks(s) or CRLM (AUC = 95.9 vs. 97.7 [p = 0.039] and AUC = 96.5 vs. 93.3 [p = 0.026], respectively), but not when scoring 3 versus 2 blocks or CRLM (both p > 0.2). After two training sessions the interobserver agreement for both the pathologist and the PhD candidate were excellent (k = 0.953 and k = 0.951, respectively). The histopathological growth patterns of colorectal liver metastasis exhibit little heterogeneity and can be determined with a high diagnostic accuracy, making them a reliable and replicable histological biomarker.

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.

Adolescent girls and young women (AGYW) remain underserved and at risk for HIV acquisition in Ethiopia. However, there is significant risk heterogeneity among AGYW with limited consensus on optimal strategies of identifying vulnerable AGYW. This study assessed the utility of venue-based sampling approaches to identify AGYW at increased risk for HIV infection. Venue mapping and time-location-sampling (TLS) methods were used to recruit AGYW from three sub-cities of Addis Ababa, February-June 2018. Interviewer-administered surveys captured socio-demographic and behavioral characteristics. Measures of AGYW vulnerability were assessed geographically and described by venue type. A total of 2468 unique venues were identified, of which 802 (32%) were systematically selected for validation and 371 (46%) were eligible including many sites that would traditionally not be included as venues in need of HIV prevention services. Overall, 800 AGYW were enrolled across 81 sampled venues. AGYW reached were largely out-of-school (n = 599, 75%) with high proportions of AGYW reporting transactional sex (n = 101, 12.6%), food insecurity (n = 165, 20.7%) and migration (n = 565, 70.6%). Taken together, these data suggest the utility of TLS methods in reaching vulnerable, out-of-school AGYW in Addis Ababa, Ethiopia.