A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

CRISPR/Cas9-based tissue-specific knockout techniques are essential for probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of single guide (sg)RNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 genes restores neutrophil motility in the corresponding knockout background. Moreover, active Rac and force-bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.

Epigenetic Silencing of Recombinant Adeno-associated Virus Genomes by NP220 and the HUSH Complex.

The single-stranded DNA genome of adeno-associated viruses (AAV) undergoes second-strand synthesis and transcription in the host cell nucleus. While wild-type AAV genomes are naturally silenced upon integration into the host genome, recombinant AAV (rAAV) genomes typically provide robust expression of transgenes persisting as extrachromosomal DNA or episomes. Episomal DNA associating with host histones is subject to epigenetic modifications, although the mechanisms underlying such are not well understood. Here, we provide evidence that the double-stranded DNA binding protein NP220, in association with the human silencing hub (HUSH) complex, mediates transcriptional silencing of single-stranded as well as self-complementary rAAV genomes. In cells lacking NP220 or other components of the HUSH complex, AAV genome transcript levels are increased and correlate with a marked reduction in repressive H3K9 histone methylation marks. We also provide evidence that the AAV capsid (serotype) can profoundly influence NP220-mediated silencing of packaged genomes, indicating potential role(s) for capsid-genome or capsid-host factor interactions in regulating epigenetic silencing of rAAV genomes. IMPORTANCE Recombinant AAV vectors can enable long-term gene expression in a wide variety of tissues. However, transgene silencing has been reported in some human gene therapy clinical trials. Here, we demonstrate the HUSH complex can suppress transcript formation from rAAV vector genomes by epigenetic modification of associated host histones. Further, the AAV capsid appears to play an important role in this pathway. We postulate that modulation of epigenetic pathways could help improve rAAV expression.

Interception of host angiogenic signalling limits mycobacterial growth.

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol.

Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches.

Effect of Lesion Location on Prostate Cancer Detection Rate with Magnetic Resonance Imaging Targeted Biopsy in African Americans.

PURPOSE: The updated PI-RADS™ (Prostate Imaging Reporting and Data System) version 2 defines different grading parameters for lesions located in the peripheral zone vs the transition zone. It has contributed to the implementation of magnetic resonance imaging targeted biopsy. In this study we evaluated the efficacy of magnetic resonance imaging targeted biopsy among African American patients with additional consideration for lesion location on magnetic resonance imaging. MATERIALS AND METHODS: We performed a retrospective review of magnetic resonance imaging targeted biopsy at a single institution where a racially diverse population is treated. A single radiology group read the prostate multiparametric magnetic resonance imaging scans and followed PI-RADS version 2 algorithms to categorize lesions. RESULTS: A total of 214 lesions from 125 men were included in the analysis, of which 162 (75.7%) were in the peripheral zone and 52 (24.3%) were in the transition zone. There were 64 lesions from African American patients and 150 from Caucasian patients with tumor location distributed proportionately. The 48 anterior lesions (22.4%) had a higher PI-RADS version 2 score and trended toward a larger size. The overall cancer detection rate was 50%, which did not differ significantly between prostate zones (p = 0.5468) or racial groups (p = 0.2294). The cancer upgrade rate was 41% and it also did not differ significantly between prostate zones (p = 0.5134) or racial groups (p = 0.2365). Anterior lesions had a higher cancer detection rate (p = 0.0117) and trended toward a higher cancer upgrade rate (p = 0.0781). CONCLUSIONS: This study provides evidence that magnetic resonance imaging targeted biopsy is equally effective in African American and Caucasian men, and does not preferentially identify prostate cancer in the peripheral zone or the transition zone.

Identification of the mating-type (MAT) locus that controls sexual reproduction of Blastomyces dermatitidis.

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

Impact of Anti-Discrimination Legislation on Access to Gender-affirming Care: A Commercial Claims Analysis.

OBJECTIVE: To examine the temporal relationship between the anti-discrimination rules of the Affordable Care Act, which took full effect in 2017, and the incidence of commercial claims for gender-affirming care, as well as cost sharing for these services. METHODS: We used a previously described algorithm to define a cohort of gender-diverse adults in the MarketScan Commercial Claims and Encounters Database. Claims for gender-affirming medical and surgical care were identified using International Classification of Diseases and Current Procedural Terminology codes plus pharmacy data; the annual incidence of surgical claims was calculated. Interrupted time series analyses were used to evaluate the temporal relationship between claims and anti-discrimination legislation. Claims data were also used to evaluate the patient contribution towards services. RESULTS: There were 70,733 gender-diverse adults included in the study and 36,702 (51.9%) of them filed claims for gender-affirming care. The incidence of persons with claims for gender-affirming surgery increased from 0.002% in 2009 to 0.012% in 2021. Interrupted time series analyses demonstrated a greater year-to-year increase in claims after anti-discrimination policy influences took effect. This change was greatest for transmasculine chest procedures. The median lifetime net payment for gender-affirming surgery was $12,429.10 and cost sharing was $1019.20 (8.6%). CONCLUSION: Commercial claims for gender-affirming surgery increased temporally with respect to implementation of anti-discrimination legislation and cost-sharing was reasonably low. However, many gender-diverse persons did not have claims for gender-affirming care, which may indicate continued out-of-pocket payment for these services.

Family Planning and Fertility Counseling Perspectives of Gender Diverse Adults and Youth Pursuing or Receiving Gender Affirming Hormone Therapy.

OBJECTIVE: To describe family planning and fertility counseling perspectives of reproductive-age gender diverse adults and youth pursuing gender affirming hormone therapy. MATERIALS AND METHODS: This was a cross sectional survey study of gender diverse adults and youth pursuing or receiving gender affirming hormone therapy. The primary outcomes of interest were parental desire and priorities for fertility preservation. RESULTS: Fifty-seven individuals (46 adults and 11 youths) completed the survey; 51% were transgender women, 35% were transgender men, and 14% identified as non-binary. 32 participants expressed interest in (n = 15, 26%) or uncertainty about (n = 18, 32%) future parenthood. 48% of participants had considered gamete cryopreservation, but only 7% each previously completed or planned to pursue this fertility option; 67% cited cost as a barrier. Participants with interest in or uncertainty about future parenthood were more likely to consider cryopreservation (P <.001) or stopping hormones for fertility preservation (P <.001). 58% of respondents reported discussing fertility preservation with a health care provider with lower rates among youth participants (P = .017). From a family planning perspective, 58% of respondents described counseling as adequate; 23% described it as inadequate and 19% reported not receiving any counseling. Participants who endorsed strong or uncertain parental desire were more likely to report inadequate counseling (P = .016). CONCLUSION: Gender diverse individuals interested in or undecided about future parenthood were more likely to consider cryopreservation and report inadequate family planning counseling. Therefore, current counseling practices may be insufficient and referral to a fertility specialist should be considered.

Conservation and divergence of four kiwifruit SVP-like MADS-box genes suggest distinct roles in kiwifruit bud dormancy and flowering.

MADS-box genes similar to Arabidopsis SHORT VEGETATIVE PHASE (SVP) have been implicated in the regulation of flowering in annual species and bud dormancy in perennial species. Kiwifruit (Actinidia spp.) are woody perennial vines where bud dormancy and out-growth affect flower development. To determine the role of SVP-like genes in dormancy and flowering of kiwifruit, four MADS-box genes with homology to Arabidopsis SVP, designated SVP1, SVP2, SVP3, and SVP4, have been identified and analysed in kiwifruit and functionally characterized in Arabidopsis. Phylogenetic analysis indicate that these genes fall into different sub-clades within the SVP-like gene group, suggesting distinct functions. Expression was generally confined to vegetative tissues, and increased transcript accumulation in shoot buds over the winter period suggests a role for these genes in bud dormancy. Down-regulation before flower differentiation indicate possible roles as floral repressors. Over-expression and complementation studies in Arabidopsis resulted in a range of floral reversion phenotypes arising from interactions with Arabidopsis MADS-box proteins, but only SVP1 and SVP3 were able to complement the svp mutant. These results suggest that the kiwifruit SVP-like genes may have distinct roles during bud dormancy and flowering.

Cost of Intralesional Collagenase Clostridium Histiolyticum Therapy Versus Surgery for the Management of Peyronie's Disease: A Claims-Based Analysis (2009-2019).

BACKGROUND: Collagenase Clostridium histolyticum (CCH), which was approved by the FDA for the treatment of Peyronie's disease (PD) in 2013, may obviate the need for surgery but its historically high cost must be considered when offering CCH vs surgical intervention to affected patients. AIM: To compare trends of intralesional injections vs surgical treatment for PD and assess the contemporary cost of treatment with CCH vs surgical intervention. METHODS: We reviewed 2009-2019 MarketScan Commercial Claims data to identify all men 18 years and older with PD. CPT and HCPCS codes were used to identify PD treatments for each patient. Associated insurance claims in USD were summed for each treatment type. OUTCOMES: Total and out-of-pocket costs, as well as frequencies, for treatments were calculated on a yearly basis and the Cochran-Armitage test was used to compare frequencies before and after FDA approval of CCH. RESULTS: Of 89,205 men diagnosed with PD, 21,605 (24.2%) underwent treatment; most required only intralesional injections, however 1,519 (7.0%) received only surgical therapy and 1,951 (9.0%) required medical and surgical therapy. Intralesional CCH use sharply increased after its FDA-approval in 2013 with a concomitant fall of intralesional verapamil use. The use of both surgical plication and plaque grafting decreased steadily from 2009 to 2019. The median cost per patient for all 3 treatments increased over the study time-period: $1,856 to $3,196 for plication, $2,233 to $3,631 for plaque grafting, and $6,940 to $8,895 per cycle for CCH. Out-of-pocket median patient contribution for plication, plaque grafting, and per cycle intralesional CCH injection were similar over the study period and never exceeded $300. CLINICAL IMPLICATIONS: CCH is significantly more expensive than any surgical treatment option, however, the out-of-pocket patient contribution for surgery and CCH are similar. STRENGTHS & LIMITATIONS: This study incorporated all procedure costs and is the most contemporary, comprehensive, and accurate reflection of overall and out-of-pocket costs to patients for surgical and intralesional PD therapies. We anticipate these data to allow for a more complete discussion between patients and providers regarding their care. The use of a commercial claims database prohibited assessment of post-procedural costs and treatment outcomes. CONCLUSION: CCH use has increased significantly since its FDA approval in 2013 with out-of-pocket patient contribution comparable to surgical therapy despite significantly higher total treatment costs. Walton EL, Quinn TP, Mulloy E, et al. Cost of Intralesional Collagenase Clostridium Histiolyticum Therapy Versus Surgery for the Management of Peyronie's Disease: A Claims-Based Analysis (2009-2019). Sex Med 2022;10:100517.

Macrophage NFATC2 mediates angiogenic signaling during mycobacterial infection.

During mycobacterial infections, pathogenic mycobacteria manipulate both host immune and stromal cells to establish and maintain a productive infection. In humans, non-human primates, and zebrafish models of infection, pathogenic mycobacteria produce and modify the specialized lipid trehalose 6,6'-dimycolate (TDM) in the bacterial cell envelope to drive host angiogenesis toward the site of forming granulomas, leading to enhanced bacterial growth. Here, we use the zebrafish-Mycobacterium marinum infection model to define the signaling basis of the host angiogenic response. Through intravital imaging and cell-restricted peptide-mediated inhibition, we identify macrophage-specific activation of NFAT signaling as essential to TDM-mediated angiogenesis in vivo. Exposure of cultured human cells to Mycobacterium tuberculosis results in robust induction of VEGFA, which is dependent on a signaling pathway downstream of host TDM detection and culminates in NFATC2 activation. As granuloma-associated angiogenesis is known to serve bacterial-beneficial roles, these findings identify potential host targets to improve tuberculosis disease outcomes.

Vaginal cuff pyoderma gangrenosum with associated ureteral stricture: A case report.

Pyoderma gangrenosum is a sterile inflammatory disease of unknown etiology characterized by recurrent cutaneous ulcers. It can occur in extracutaneous locations, especially at operative sites, and has been reported following gynecologic surgery. This report is the first case of pyoderma gangrenosum as a remote complication of pelvic surgery with associated ureteral stricture. It demonstrates the diagnostic challenge of this rare disease and the importance of broadening the differential diagnosis when apparent infections do not respond to treatment to minimize the morbidity of ineffective antibiotic and surgical interventions.

An FTIR study of the induction and release of kiwifruit buds from dormancy.

BACKGROUND: Many deciduous, perennial fruit crops require winter chilling for adequate budbreak and flowering. Recent research has shown that changes in sugar and amino acid profiles are associated with the release of buds from dormancy. This paper uses FTIR spectrometry to provide an alternative mechanism for tracking metabolic changes in the meristems of kiwifruit buds during winter dormancy. The results suggest that the application of multivariate analysis to FTIR spectra has the potential to be a reliable and fast method for detecting structural and compositional changes in fruit crops. RESULTS: Ten wave numbers of the FTIR spectra are used to calculate a bud development function. This function has been validated using data from two seasons and four orchards, and by monitoring the effects of hydrogen cyanamide application, sugar concentrations and soil temperatures on this function. These wave numbers appear to be associated with carbohydrate, pectin and cellulose levels in the meristems. CONCLUSION: It is expected that this FTIR signature can be used to advance our understanding of the influence of the various environmental and physiological factors on the breaking of bud dormancy and shoot outgrowth, including the optimum timing and concentrations of applications of budbreak regulators, such as hydrogen cyanamide.

A rapid transcriptional activation is induced by the dormancy-breaking chemical hydrogen cyanamide in kiwifruit (Actinidia deliciosa) buds.

Budbreak in kiwifruit (Actinidia deliciosa) can be poor in locations that have warm winters with insufficient winter chilling. Kiwifruit vines are often treated with the dormancy-breaking chemical hydrogen cyanamide (HC) to increase and synchronize budbreak. This treatment also offers a tool to understand the processes involved in budbreak. A genomics approach is presented here to increase our understanding of budbreak in kiwifruit. Most genes identified following HC application appear to be associated with responses to stress, but a number of genes appear to be associated with the reactivation of growth. Three patterns of gene expression were identified: Profile 1, an HC-induced transient activation; Profile 2, an HC-induced transient activation followed by a growth-related activation; and Profile 3, HC- and growth-repressed. One group of genes that was rapidly up-regulated in response to HC was the glutathione S-transferase (GST) class of genes, which have been associated with stress and signalling. Previous budbreak studies, in three other species, also report up-regulated GST expression. Phylogenetic analysis of these GSTs showed that they clustered into two sub-clades, suggesting a strong correlation between their expression and budbreak across species.

Use of Disposable Punch Biopsy Device to Add Foley Catheter Fenestration to Improve Drainage of Post Radical Prostatectomy Anastomotic Leak.

CONTEXT: Radical prostatectomy (RP) is a major oncologic urological surgery that can have high morbidity if complications arise. Bladder-urethral urine anastomotic leaks (AL) are one of the most common complications and can greatly increase morbidity. To date, there are few resources to manage AL. One management technique is using a Foley catheter with an additional auxiliary drainage port, also known as a fenestrated catheter. This type of auxiliary drainage port allows a low-pressure drainage source that is located near the anastomosis to increase urine drainage from catheter rather than from the AL site. The optimal size and location of this additional drainage port is currently unknown. This experiment evaluated the optimal auxiliary drainage port size and an inexpensive technique to easily construct such a catheter. METHODS: Utilizing different size punch biopsies, auxiliary drainage ports were placed in different size Foley catheters and drainage rates and the structural integrity of the catheter was assessed. RESULTS: A 3.0 mm punch biopsy located 1.0 cm proximal to the Foley balloon in an 18 French (Fr) catheter was determined to be the optimal size. A 2.0 mm punch biopsy provided significantly less drainage. The 4.0 mm punch biopsy compromised the structural integrity of the catheter. CONCLUSIONS: Based on these experimental results, we recommend using a 3.0 mm punch biopsy in an 18 Fr catheter 1.0 cm. proximal to the balloon for an auxiliary drain site in Foley catheter when the anastomosis is not watertight or the surgeon has reason to believe the patient is at higher risk for an AL Factors such as history of pelvic radiation, abnormal anatomy, large prostate, post-surgical hematoma formation, obesity, previous prostatic surgery, difficult anastomosis, blood loss and postoperative urinary tract infection may make use of this type of device more attractive.

Prostate volume measured by magnetic resonance imaging is not a predictor of lower urinary tract symptoms.

PURPOSE: Prostate volume is frequently utilized to counsel patients presenting to family medicine physicians with voiding complaints. We evaluated the relation between International Prostate Symptom Score (IPSS) and prostate volume measured by phased-array surface coil magnetic resonance imaging (MRI). METHODS: We performed an institutional review board (IRB)-approved retrospective study of all patients who received a prostate MRI between 2015 and 2017. Correlation between the overall IPSS, IPSS components, prostate volume stratified by prostate specific antigen (PSA) (<1.4 vs. ≥1.4 g/dL), and race (black vs. white) was examined. RESULTS: In all, 592 patients had prostate MRIs performed between 2015 and 2017. Two hundred and twenty-nine of these patients had IPSS and prostate volume information available in their medical records. The mean age of the cohort was 64.67 (SD = ±7.82) and mean PSA was 7.75 (SD = ±8.3). The mean IPSS was 9.77 (SD ± 7.2), and mean prostate volume was 55.88 cubic cm (SD = ±38.9). The correlation coefficient between prostate volume and IPSS was 0.12789 (P = 0.05). The correlation between prostate volume and IPSS was also not significant in 128 men with prostate volume above 40 cubic cm. Stratifying analysis by race and PSA showed no significant correlation between volume and IPSS. Analysis of the correlation between the different dimension of prostate volume and IPSS revealed significant but weak associations. CONCLUSIONS: Even with more precise estimation with MRI, prostate volume does not predict obstruction complaints. This finding is of importance when treating males presenting with voiding dysfunction to primary care.

Barriers to obtaining prostate multi-parametric magnetic resonance imaging in African-American men on active surveillance for prostate cancer.

PURPOSE: Magnetic resonance imaging is playing an ever-bigger role in the management of prostate cancer. This study investigated barriers to obtaining multi-parametric MRI (mpMRI) in African-American men on active surveillance for prostate cancer in comparison to white men affected by the same type of cancer. MATERIALS AND METHODS: Retrospective review of prostate mpMRI orders from August 2015 to October 2017 at a single health organization treating a diverse population was performed. Data was extracted from the electronic medical records and cancellations were examined based on the documented reason for mpMRI cancellation, race, median zip code household income, and distance from healthcare facility. RESULTS: Out of 793 prostate mpMRI orders, 201 (25%) went unscanned. Access to care issues accounted for 46% of unscanned orders. Patient cancellations were the most common, followed by difficulty contacting patients, and insurance denials. African-American patients disproportionately went unscanned because institution staff were unable to contact patients (29% vs 10% in white men, P = 0.0015). Median zip code household income was significantly different between racial groups but did not vary between indication for cancellation. CONCLUSIONS: African-American prostate cancer patients' access to mpMRI is hindered more by barriers to care than White patients. Urology providers must consider these issues before using prostate mpMRI within their active surveillance pathways.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling.

Mycobacterial infection leads to the formation of characteristic immune aggregates called granulomas, a process accompanied by dramatic remodeling of the host vasculature. As granuloma angiogenesis favors the infecting mycobacteria, it may be actively promoted by bacterial determinants during infection. Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. cis-Cyclopropanation of mycobacterial mycolic acids by pcaA drives the activation of host Vegf signaling within granuloma macrophages. Cyclopropanation of the mycobacterial cell wall glycolipid trehalose dimycolate is both required and sufficient to induce robust host angiogenesis. Inducible genetic inhibition of angiogenesis and Vegf signaling during granuloma formation results in bacterial growth deficits. Together, these data reveal a mechanism by which PcaA-mediated cis-cyclopropanation of mycolic acids promotes bacterial growth and dissemination in vivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.