Evaluation of the Efficacy and Safety of Pedicled Perforator Flap Technique for Salvage Nipple Reconstruction in Breast Cancer Patients: A Pilot Study.

OBJECTIVE: We propose a pedicled perforator flap technique for salvage nipple reconstruction after initial nipple reconstruction fails in breast cancer patients. METHODS: This is a pilot study. A total of 21 female breast cancer patients who underwent nipple reconstruction following initial nipple reconstruction fails were enrolled, and salvage nipple reconstruction based pedicled perforator flap were performed between 2016 and 2020. Operative time, perforator design, postoperative complications, follow-up duration, projection of nipple, as well as patient-reported outcomes measured by the BREAST-Q and visual analogue scale (VAS) were assessed. RESULTS: Sixteen patients underwent fifth lateral intercostal artery perforator reconstruction, while 5 patients underwent fifth anterior intercostal artery perforator flap reconstruction. The surgeries were successful without intraoperative complications, with a mean operative time of 67 minutes. Postoperative complications were absent. The mean follow-up duration was 18 months. The mean nipple projection was 8 mm (range, 6-10 mm) with a shrinkage of 20% at 6 months after surgery. The average scores for psychosocial well-being, satisfaction with breasts, and satisfaction with nipples domains of the BREAST-Q significantly increased (P < .01) at 6 months post-reconstruction. Sexual well-being subdomain showed no statistical difference (P = .9369). The VAS scores for cosmesis and patient satisfaction with surgery were 9 and 9.3, respectively. CONCLUSION: The pedicled perforator flap technique for salvage nipple reconstruction is a safe and effective approach.

Predicting immunotherapy response in melanoma using a novel tumor immunological phenotype-related gene index.

Introduction: Melanoma is a highly aggressive and recurrent form of skin cancer, posing challenges in prognosis and therapy prediction. Methods: In this study, we developed a novel TIPRGPI consisting of 20 genes using Univariate Cox regression and the LASSO algorithm. The high and low-risk groups based on TIPRGPI exhibited distinct mutation profiles, hallmark pathways, and immune cell infiltration in the tumor microenvironment. Results: Notably, significant differences in tumor immunogenicity and TIDE were observed between the risk groups, suggesting a better response to immune checkpoint blockade therapy in the low-TIPRGPI group. Additionally, molecular docking predicted 10 potential drugs that bind to the core target, PTPRC, of the TIPRGPI signature. Discussion: Our findings highlight the reliability of TIPRGPI as a prognostic signature and its potential application in risk classification, immunotherapy response prediction, and drug candidate identification for melanoma treatment. The "TIP genes" guided strategy presented in this study may have implications beyond melanoma and could be applied to other cancer types.

A Fully Automated Mini-Mental State Examination Assessment Model Using Computer Algorithms for Cognitive Screening.

Background: Rapidly growing healthcare demand associated with global population aging has spurred the development of new digital tools for the assessment of cognitive performance in older adults. Objective: To develop a fully automated Mini-Mental State Examination (MMSE) assessment model and validate the model's rating consistency. Methods: The Automated Assessment Model for MMSE (AAM-MMSE) was an about 10-min computerized cognitive screening tool containing the same questions as the traditional paper-based Chinese MMSE. The validity of the AAM-MMSE was assessed in term of the consistency between the AAM-MMSE rating and physician rating. Results: A total of 427 participants were recruited for this study. The average age of these participants was 60.6 years old (ranging from 19 to 104 years old). According to the intraclass correlation coefficient (ICC), the interrater reliability between physicians and the AAM-MMSE for the full MMSE scale AAM-MMSE was high [ICC (2,1)=0.952; with its 95% CI of (0.883,0.974)]. According to the weighted kappa coefficients results the interrater agreement level for audio-related items showed high, but for items "Reading and obey", "Three-stage command", and "Writing complete sentence" were slight to fair. The AAM-MMSE rating accuracy was 87%. A Bland-Altman plot showed that the bias between the two total scores was 1.48 points with the upper and lower limits of agreement equal to 6.23 points and -3.26 points. Conclusions: Our work offers a promising fully automated MMSE assessment system for cognitive screening with pretty good accuracy.

A machine learning-based approach to assess impacts of autonomous vehicles on pavement roughness.

Studies have been initiated to investigate the potential impact of connected and automated vehicles (CAVs) on transportation infrastructure. However, most existing research only focuses on the wandering patterns of CAVs. To bridge this gap, an apple-to-apple comparison is first performed to systematically reveal the behavioural differences between the human-driven vehicle (HDV) and CAV trajectory patterns for the first time, with the data collected from the camera-based next generation simulation dataset and autonomous driving co-simulation platform, CARLA and SUMO, respectively. A gradient boosting-based ensemble learning model for pavement performance (i.e. international roughness index) prediction is then developed with the input features including three driving pattern features, namely, lateral wandering deviation, longitudinal car-following distance and driving speed, plus 20 other context variables. A total of 1707 observations is extracted from the long-term pavement performance database for model training purposes. The result indicates that the trained model can accurately predict pavement deterioration and that CAV deteriorates pavement faster than HDV by 8.1% on average. According to the sensitivity analysis, CAV deployment will create a greater impact on the younger pavements, and the rate of pavement deterioration is found to be stable under light traffic, whereas it will increase under congested traffic. This article is part of the theme issue 'Artificial intelligence in failure analysis of transportation infrastructure and materials'.

Neonatal exposure of bisphenol A led to increased lipolysis of visceral adipose tissue in adult rats with DNA hypomethylation of Atgl being one of the possible underlying mechanisms.

Accumulating evidence suggests the role of developmental exposure of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism remains unclear. Using a rat model, we investigated the neonatal exposure of BPA on lipid metabolism in adult and the underlying mechanisms. From postnatal day1(PND1) to PND10, male rats were exposed to BPA via daily subcutaneous injection with 10 µg/100 µL BPA (1.24-0.5 mg/kg body weight/day, a dose below the US-EPA LOAEL). After fasting for 8 h, adult rats aged 80 days showed elevated levels of serum free fatty acid (FFA), glycerol and glucose, and increased levels of FFA and glycerol in visceral adipose tissue. The expression levels of key enzymes of lipolysis, adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl), were increased in visceral adipose tissue from BPA-exposed rats after fasting. On the other hand, transcription levels of lipogenic genes remained unchanged. Differentiation of visceral adipocyte in rats takes place neonatally. In our study, neonatal BPA exposure induced DNA hypomethylation of Atgl in visceral adipose tissue. In 3T3-L1 cell, administration of 10-7 mol/L BPA throughout the differentiation stage led to DNA hypomethylation and increased expression of Atgl. Our results suggest that neonatal exposure of BPA led to increased lipolysis of visceral adipose tissue in young adults, which will predispose individuals to multiple metabolic disorders. The DNA hypomethylation of Atgl might be one of the mechanisms underneath the long-lasting effect of neonatal BPA exposure.

Social-economic impact of infection after cosmetic facial injection: A retrospective study.

BACKGROUND: The overwhelming interest in economically cost-effective treatments had led to an increase in the development of cosmetic facial injections. Concomitantly, complications have been also on the increasing trend. This article aimed to investigate the social-economic impact caused by infection after cosmetic facial injection. METHODS: A retrospective study was conducted between January 2014 and December 2019. Information including demographics, location of the original injection site, treatments, economic loss, and time away from work associated with complications was documented. Patients were asked to complete 2 FACE-Q domains 4 weeks after the first presentation, including quality of life and patient experience. RESULTS: Complete data were available for 12 patients. All patients were female; the mean age was 32.66 years (range, 26-49 years). The mean therapeutic cost of atypical mycobacterium was 18170.58 USD. The mean time away from work was 51.25 days (range, 21-103 days). The mean cost of time away from work was 400.80 USD (range, 164.23-805.50 USD). From FACE-Q, patients reported a decreased quality of social life, including social function (38.83 ± 12.10), psychological distress (48.67 ± 11.01), and early-life impact (39.92 ± 10.15). Patients also reported low satisfaction with their decision to undergo the cosmetic facial injection. CONCLUSIONS: Infection after cosmetic facial injection comes at a considerable social-economic impact on patients. Patients who responded in this study were extremely unsatisfied with the quality of life following the infection.

Pyroptosis-Related lncRNAs for Predicting the Prognosis and Identifying Immune Microenvironment Infiltration in Breast Cancer Lung Metastasis.

Breast cancer (BC) is the second leading cause of death among women and is highly heterogeneous. Three pyroptosis (PR) subtypes were identified in patients with BC from the Cancer Genome Atlas Database (TCGA) cohorts using 20 PR-related regulators, which illustrate a strong association between BC prognosis and PR. Lung metastasis commonly occurs in the advanced stages of BC, resulting in a poor quality of life. Eight differentially expressed (DE) lncRNAs were identified using LASSO-Cox analysis between PR-related and BC lung metastasis. Moreover, a BRCA risk-score (RS) model was established using multivariate Cox regression, which correlated with prognosis in TCGA-BRCA. Clinical characteristics, tumor mutation burden, and tumor immune cell infiltration were extensively investigated between high- and low-RS groups. Similarly, a lower RS implied longer overall survival, greater inflammatory cell infiltration, and better immunotherapeutic response to PD-1 blockers. Our findings provide a foundation for future studies targeting PR and confirme that RS could predict the prognosis of patients with BC.

The psychological impact on patients with memory disorders and their caregivers during COVID-19.

BACKGROUND: COVID-19 is erupting globally. Mass quarantine had been implemented all around China which could influence the psychological status of patients with memory disorders and their caregivers. AIM: To investigate the psychological impact of mass quarantine on patients with memory disorders and their caregivers in China. METHODS: We completed a cross-sectional study in 787 patients and their caregivers registered from 2010 to 2019 in Memory Clinic, The First Affiliated Hospital of Chongqing Medical University, by telephone interviews. The performance in neuropsychiatric symptoms (NPSs), sleep, nutrition, chronic diseases of patients, and the burden of care, anxiety and depression of caregivers was assessed by six assessment scales (MNA-SF, PSQI, NPI, RSS, PHQ-9 and GAD-7). RESULTS: Only 68 (8.6%) patients worried about the outbreak of COVID-19. The prevalence of NPSs among all subjects was nearly 60.0%. Approximately 50.0% of the caregivers reported distress. More than 70.0% of patients remained stable in NPSs. However, anxiety, depression, aberrant motor disorder and delusion were exacerbated (22.9%, 18.6%, 17.1% and 16.8%, respectively). Appetite and eating disorder led alleviation rate by 25.8% while disappearing rate of agitation led by 5.8%. 7.5% of caregivers manifested depressive symptoms while 4.9% expressed anxiety symptoms, and 40.8% showed care burden. The coefficients of RSS and PHQ-9, RSS and GAD-7, RSS and NPI-D, PHQ-9 and GAD-7 were 0.7, 0.5, 0.5 and 0.6, respectively (p < 0.01). CONCLUSION: Changes in NPSs during COVID-19 were observed in some patients with memory disorders and their caregivers, and adherence to medication contributed to the stabilization of NPSs.

GPR30-mediated HMGB1 upregulation in CAFs induces autophagy and tamoxifen resistance in ERα-positive breast cancer cells.

Tamoxifen (TAM) resistance constitutes a challenge in managing estrogen receptor (ER)α+ breast cancer patients. G-protein-coupled estrogen receptor (GPR30/GPER), which reportedly initiates TAM resistance in ERα+/ GPR30+ breast cancers, is detected in the breast cancer microenvironment, especially cancer associated fibroblasts (CAFs). Herein, considering that GPR30 mediates transcriptional regulation in different cell backgrounds, a microarray strategy was applied in immortalized CAFs derived from primary breast cancer samples, resulting in the identification of 165 GPR30 target genes, among which HMGB1 was confirmed to be upregulated by 17-ß estradiol(E2)- and TAM-triggered GPR30 activation in CAFs. Activated GPR30 increased extracellular HMGB1 secretion by CAFs, which was reduced by blocking PI3K/AKT signaling using G15 or LY294002. GPR30-induced HMGB1 upregulation triggered MEK/ERK signaling, leading to increased autophagic behavior to protect cancer cells from TAM-induced apoptosis, mimicking the recombinant HMGB1-mediated increase in cancer cell resistance potential to TAM. MEK/ERK signaling blockage by U0126 decreased the autophagic behavior and resistance ability of cancer cells to TAM. CAF-expressed GPR30 induced TAM resistance via HMGB1 in vivo. Overall, TAM upregulated HMGB1 expression and secretion in CAFs via GPR30/PI3K/AKT signaling, and the secreted HMGB1 induced autophagy to enhance TAM resistance in MCF-7 cells in an ERK-dependent manner. Thus, targeting GPR30 and downstream cascades may be an effective strategy to attenuate the resistance of ERα-positive breast tumors to endocrine therapy.

The Neuropsychiatric Changes After COVID-19 Quarantine in Patients With Cognitive Impairment and Their Caregivers in Chongqing, China: A Cohort Study.

BACKGROUND: The follow-up study on neuropsychiatric changes after the lifting of coronavirus disease 2019 (COVID-19) quarantine in patients with cognitive impairment and their caregivers is still lacking, and relative information is needed to formulate more comprehensive healthcare prevention measures worldwide. AIMS: To provide data on the changes in neuropsychiatric performance after the lifting of COVID-19 quarantine in patients with cognitive disorders and their caregivers. METHODS: Two surveys in Chongqing, China were conducted via telephonic interview with 531 patients and their caregivers. The baseline survey was performed from February 11 to 23, 2020, and the follow-up was from October 24 to November 9, 2020. The data of neuropsychiatric symptoms (NPSs), sleep, nutrition, and chronic diseases of patients, as well as the burden of care, anxiety, and depression of caregivers were evaluated. RESULTS: Significant alleviation of NPSs after the lifting of COVID-19 quarantine was observed in patients with mild cognitive impairment (MCI) and dementia (both P < 0.05). Compared with baseline, the prevalence for NPSs of all participants dropped from 57.94 to 38.82%. Among NPS subdomains, apathy displayed the biggest decline at follow-up by 10.72%, followed by nighttime behavior by 8.65%. Mixed effect generalized estimation equation analysis showed significant amelioration in hallucination, depression, apathy, irritability, aberrant motor behavior, and nighttime behavior (all P < 0.05), with the most prominent changes in nighttime behavior and apathy. Among the patients with unsatisfactory control of chronic disease, the medication adherence rate dropped by approximately 30% after the lifting of quarantine. More importantly, around 13% increase of care burden was observed among the caregivers at follow-up, with both depression and anxiety rising by nearly 4%. CONCLUSION: The prolonged quarantine may exacerbate NPS in patients with memory disorders, while the care burden and mental stability of the caregivers after the pandemic should also be concerned.

Associations between malnutrition and cognitive impairment in an elderly Chinese population: an analysis based on a 7-year database.

AIM: This study aimed to elucidate whether malnutrition is associated with cognitive impairment in an older Chinese population. METHODS: A cross-sectional study was conducted in 2365 participants aged 60 years or older from January 2013 to September 2019. Nutritional status was measured by using the Mini Nutritional Assessment Short Form (MNA-SF). Cognitive function was assessed with the Mini-Mental State Examination (MMSE). The relationship between malnutrition or each Mini Nutritional Assessment Short Form domain and cognitive impairment was examined with univariate and multivariate logistic regression analysis. RESULTS: The prevalence of malnutrition, risk of malnutrition, and cognitive impairment was 5.54%, 33.45%, and 36.74%, respectively. The prevalence was higher in those 80 years and older: 7.88%, 40.75%, and 53.65%, respectively. The Mini-Mental State Examination score was positively correlated with the Mini Nutritional Assessment Short Form score (r = 0.364, P < 0.001). After adjustment for age, gender, education, marital status, and living alone, malnutrition (odds ratio (OR) = 3.927, 95% confidence interval (CI): 2.650-5.819), anorexia (OR = 1.454, 95%CI: 1.192-1.774), weight loss (OR = 1.697, 95%CI: 1.406-2.047), impaired mobility (OR = 4.156, 95%CI: 3.311-5.218), and psychological stress (OR = 1.414, 95%CI: 1.070-1.869) were significantly associated with an increased risk of cognitive impairment. CONCLUSIONS: Our results suggest that the prevalence of malnutrition and cognitive impairment is relatively high and increases with age. Malnutrition, anorexia, weight loss, impaired mobility, and psychological stress are significantly associated with an increased risk of cognitive impairment. Therefore, clinicians should assess the nutritional and cognitive status of the elderly regularly to improve early detection and timely intervention.

Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma.

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl2 ) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.

Association between TERT rs2853669 polymorphism and cancer risk: A meta-analysis of 9,157 cases and 11,073 controls.

BACKGROUND: It has been reported that the functional telomerase reverse transcriptase (TERT) rs2853669 polymorphism might contribute to different types of human cancer. However, the association of this mutation with cancer remains controversial. Here, we conducted a meta-analysis to characterize this relationship. MATERIALS AND METHODS/MAIN RESULTS: A systematic search of studies on the association of TERT rs2853669 polymorphism with all types of cancer was conducted in PubMed, Embase and Cochrane Library. The summary odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) were used to pool the effect size in a fixed-effects model or a random-effects model where appropriate. A total of 13 articles and 15 case-control studies, including 9,157 cases and 11,073 controls, were included in this meta-analysis. Overall, the pooled results indicated that the rs2853669 polymorphism was significantly associated with increased cancer risk in a homozygote comparison model (CT vs. TT: OR = 1.085, 95% CI: 1.015-1.159, P = 0.016). In the stratified analyses, a significant increased cancer risk was observed in Asian, but not Caucasian patients. A subgroup analysis by cancer type also revealed a significant increase in the risk of lung cancer, but not breast cancer. CONCLUSIONS: The results of this meta-analysis suggest that the TERT rs2853669 polymorphism is associated with a significantly increased risk of cancer, particularly lung cancer, in Asian populations.

High throughput and accurate serum proteome profiling by integrated sample preparation technology and single-run data independent mass spectrometry analysis.

Mass spectrometry (MS)-based serum proteome analysis is extremely challenging due to its high complexity and dynamic range of protein abundances. Developing high throughput and accurate serum proteomic profiling approach capable of analyzing large cohorts is urgently needed for biomarker discovery. Herein, we report a streamlined workflow for fast and accurate proteomic profiling from 1µL of blood serum. The workflow combined an integrated technique for highly sensitive and reproducible sample preparation and a new data-independent acquisition (DIA)-based MS method. Comparing with standard data dependent acquisition (DDA) approach, the optimized DIA method doubled the number of detected peptides and proteins with better reproducibility. Without protein immunodepletion and prefractionation, the single-run DIA analysis enables quantitative profiling of over 300 proteins with 50min gradient time. The quantified proteins span more than five orders of magnitude of abundance range and contain over 50 FDA-approved disease markers. The workflow allowed us to analyze 20 serum samples per day, with about 358 protein groups per sample being identified. A proof-of-concept study on renal cell carcinoma (RCC) serum samples confirmed the feasibility of the workflow for large scale serum proteomic profiling and disease-related biomarker discovery. BIOLOGICAL SIGNIFICANCE: Blood serum or plasma is the predominant specimen for clinical proteomic studies while the analysis is extremely challenging for its high complexity. Many efforts had been made in the past for serum proteomics for maximizing protein identifications, whereas few have been concerned with throughput and reproducibility. Here, we establish a rapid, robust and high reproducible DIA-based workflow for streamlined serum proteomic profiling from 1µL serum. The workflow doesn't need protein depletion and pre-fractionation, while still being able to detect disease-relevant proteins accurately. The workflow is promising in clinical application, because the usage of small sample amounts makes blood testing much less invasive, the fully integrated sample preparation by the SISPROT technology greatly improve sample preparation throughput and reproducibility, and the scan feature of DIA method provides a way to convert nonrenewable clinical specimens into permanent digital proteome maps which could be easily reanalyzed.

ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway.

Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2 , NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors.

BACKGROUND: Rice blast caused by the fungus Magnaporthe oryzae is an important disease in virtually every rice growing region of the world, which leads to significant annual decreases of grain quality and yield. To prevent disease, resistance genes in rice have been cloned and introduced into susceptible cultivars. However, introduced resistance can often be broken within few years of release, often due to mutation of cognate avirulence genes in fungal field populations. RESULTS: To better understand the pattern of mutation of M. oryzae field isolates under natural selection forces, we used a next generation sequencing approach to analyze the genomes of two field isolates FJ81278 and HN19311, as well as the transcriptome of FJ81278. By comparing the de novo genome assemblies of the two isolates against the finished reference strain 70-15, we identified extensive polymorphisms including unique genes, SNPs (single nucleotide polymorphism) and indels, structural variations, copy number variations, and loci under strong positive selection. The 1.75 MB of isolate-specific genome content carrying 118 novel genes from FJ81278, and 0.83 MB from HN19311 were also identified. By analyzing secreted proteins carrying polymorphisms, in total 256 candidate virulence effectors were found and 6 were chosen for functional characterization. CONCLUSIONS: We provide results from genome comparison analysis showing extensive genome variation, and generated a list of M. oryzae candidate virulence effectors for functional characterization.

Histoplasma yeast and mycelial transcriptomes reveal pathogenic-phase and lineage-specific gene expression profiles.

BACKGROUND: The dimorphic fungus Histoplasma capsulatum causes respiratory and systemic disease in mammalian hosts by expression of factors that enable survival within phagocytic cells of the immune system. Histoplasma's dimorphism is distinguished by growth either as avirulent mycelia or as pathogenic yeast. Geographically distinct strains of Histoplasma differ in their relative virulence in mammalian hosts and in production of and requirement for specific virulence factors. The close similarity in the genome sequences of these diverse strains suggests that phenotypic variations result from differences in gene expression rather than gene content. To provide insight into how the transcriptional program translates into morphological variation and the pathogenic lifestyle, we compared the transcriptional profile of the pathogenic yeast phase and the non-pathogenic mycelial phase of two clinical isolates of Histoplasma. RESULTS: To overcome inaccuracies in ab initio genome annotation of the Histoplasma genome, we used RNA-seq methodology to generate gene structure models based on experimental evidence. Quantitative analyses of the sequencing reads revealed 6% to 9% of genes are differentially regulated between the two phases. RNA-seq-based mRNA quantitation was strongly correlated with gene expression levels determined by quantitative RT-PCR. Comparison of the yeast-phase transcriptomes between strains showed 7.6% of all genes have lineage-specific expression differences including genes contributing, or potentially related, to pathogenesis. GFP-transcriptional fusions and their introduction into both strain backgrounds revealed that the difference in transcriptional activity of individual genes reflects both variations in the cis- and trans-acting factors between Histoplasma strains. CONCLUSIONS: Comparison of the yeast and mycelial transcriptomes highlights genes encoding virulence factors as well as those involved in protein glycosylation, alternative metabolism, lipid remodeling, and cell wall glycanases that may contribute to Histoplasma pathogenesis. These studies lay an essential foundation for understanding how gene expression variations contribute to the strain- and phase-specific virulence differences of Histoplasma.

Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer.

BACKGROUND: Fungal plant pathogens cause serious agricultural losses worldwide. Alternaria arborescens is a major pathogen of tomato, with its virulence determined by the presence of a conditionally dispensable chromosome (CDC) carrying host-specific toxin genes. Genes encoding these toxins are well-studied, however the genomic content and organization of the CDC is not known. RESULTS: To gain a richer understanding of the molecular determinants of virulence and the evolution of pathogenicity, we performed whole genome sequencing of A. arborescens. Here we present the de-novo assembly of the CDC and its predicted gene content. Also presented is hybridization data validating the CDC assembly. Predicted genes were functionally annotated through BLAST. Gene ontology terms were assigned, and conserved domains were identified. Differences in nucleotide usage were found between CDC genes and those on the essential chromosome (EC), including GC3-content, codon usage bias, and repeat region load. Genes carrying PKS and NRPS domains were identified in clusters on the CDC and evidence supporting the origin of the CDC through horizontal transfer from an unrelated fungus was found. CONCLUSIONS: We provide evidence supporting the hypothesis that the CDC in A. arborescens was acquired through horizontal transfer, likely from an unrelated fungus. We also identified several predicted CDC genes under positive selection that may serve as candidate virulence factors.