The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

World data centre for microorganisms: an information infrastructure to explore and utilize preserved microbial strains worldwide.

The World Data Centre for Microorganisms (WDCM) was established 50 years ago as the data center of the World Federation for Culture Collections (WFCC)-Microbial Resource Center (MIRCEN). WDCM aims to provide integrated information services using big data technology for microbial resource centers and microbiologists all over the world. Here, we provide an overview of WDCM including all of its integrated services. Culture Collections Information Worldwide (CCINFO) provides metadata information on 708 culture collections from 72 countries and regions. Global Catalogue of Microorganism (GCM) gathers strain catalogue information and provides a data retrieval, analysis, and visualization system of microbial resources. Currently, GCM includes >368 000 strains from 103 culture collections in 43 countries and regions. Analyzer of Bioresource Citation (ABC) is a data mining tool extracting strain related publications, patents, nucleotide sequences and genome information from public data sources to form a knowledge base. Reference Strain Catalogue (RSC) maintains a database of strains listed in International Standards Organization (ISO) and other international or regional standards. RSC allocates a unique identifier to strains recommended for use in diagnosis and quality control, and hence serves as a valuable cross-platform reference. WDCM provides free access to all these services at www.wdcm.org.

Hepatic artery infusion of melphalan in patients with liver metastases from ocular melanoma.

BACKGROUND AND OBJECTIVES: Ocular melanoma has a predilection for liver metastases. Systemic treatment is ineffective and the optimal regional therapy approach is poorly defined. Isolated hepatic perfusion (IHP) with melphalan has emerged as a viable treatment option, however a subset of patients are not candidates for this treatment. We therefore sought to determine if melphalan could be safely administered via the hepatic artery for these patients. METHODS: A retrospective review of patients treated with hepatic artery infusion (HAI) of melphalan was undertaken. All patients had contraindications to IHP and were without other therapy options. Melphalan infusion was repeated every four weeks with consideration for dose escalation in the absence of toxicity or significant disease progression. RESULTS: Fourteen patients were treated with HAI of melphalan from 2010 to 2015. All patients had hepatic dysfunction or prohibitive tumor volume precluding IHP. There were no procedure-related complications. Three patients (21%) died within 30 days and the median survival was 2.9 months. Elevated baseline bilirubin > 2.5 mg/dL was associated with worse overall survival (0.93 vs 6.3 months, P < 0.05). CONCLUSION: HAI of melphalan is safe and feasible for patients with metastatic ocular melanoma. Further study to determine the optimal utilization of this treatment approach is warranted.

Comparative Analysis of Intranodal Lymphangiography with Percutaneous Intervention for Postsurgical Chylous Effusions.

PURPOSE: To evaluate clinical success and time to resolution of intranodal lymphangiography (INL) alone or with thoracic duct embolization (TDE) or thoracic duct disruption (TDD) based on initial effusion volume for postsurgical chylothorax. MATERIALS AND METHODS: Retrospective review was performed of 57 patients (mean age 63 y; 65% male) undergoing INL alone or in conjunction with other percutaneous techniques for postsurgical chylous effusions. INL alone was performed when chylothorax output was ≤ 500 mL/d and no leak was identified during fluoroscopy. RESULTS: INL was technically successful in all patients. There was 1 major and 2 minor complications. Clinical success rate was 71% (40/56). Clinical success rate meeting algorithmic inclusion criteria was 71.4% (5/7) for INL only, 41.7% (5/12) in INL with TDD, and 90.5% (19/21) in INL with TDE. Hazard ratio (HR) of clinical success of INL with TDE versus INL only was not statistically significant (HR 2.3, 95% confidence interval [CI], 0.70-5.87, P = .19). Median time to resolution was 14 days for INL only (95% CI, 0 days to not reached), 7 days for INL with TDD (95% CI, 4 days to not reached), and 3 days for INL with TDE (95% CI, 2 to 5 days) (P = .007). No statistically significant difference in median time to resolution existed between INL with TDE and INL only (P = .04). CONCLUSION: In patients with postsurgical chylothorax, INL alone had similar rates of clinical success and time to resolution compared with INL with TDE when initial effusion volume was ≤ 500 mL/d and no leak was visualized during fluoroscopy.

Results of a Randomized Controlled Multicenter Phase III Trial of Percutaneous Hepatic Perfusion Compared with Best Available Care for Patients with Melanoma Liver Metastases.

PURPOSE: There is no consensus for the treatment of melanoma metastatic to the liver. Percutaneous hepatic perfusion with melphalan (PHP-Mel) is a method of delivering regional chemotherapy selectively to the liver. In this study, we report the results of a multicenter, randomized controlled trial comparing PHP-Mel with best alternative care (BAC) for patients with ocular or cutaneous melanoma metastatic to the liver. PATIENTS AND METHODS: A total of 93 patients were randomized to PHP-Mel (n = 44) or BAC (n = 49). On the PHP-Mel arm, melphalan was delivered via the hepatic artery, and the hepatic effluent captured and filtered extracorporeally prior to return to the systemic circulation via a venovenous bypass circuit. PHP-Mel was repeatable every 4-8 weeks. The primary endpoint was hepatic progression-free survival (hPFS), and secondary endpoints included overall PFS (oPFS), overall survival (OS), hepatic objective response (hOR), and safety. RESULTS: hPFS was 7.0 months for PHP-Mel and 1.6 months for BAC (p < 0.0001), while oPFS was 5.4 months for PHP-Mel and 1.6 months for BAC (p < 0.0001). Median OS was not significantly different (PHP-Mel 10.6 months vs. BAC 10.0 months), likely due to crossover to PHP-Mel treatment (57.1 %) from the BAC arm, and the hOR was 36.4 % for PHP-Mel and 2.0 % for BAC (p < 0.001). The majority of adverse events were related to bone marrow suppression. Four deaths were attributed to PHP-Mel, three in the primary PHP-Mel group, and one post-crossover to PHP-Mel from BAC. CONCLUSION: This randomized, phase III study demonstrated the efficacy of the PHP-Mel procedure. hPFS, oPFS, and hOR were significantly improved with PHP-Mel. PHP with melphalan should provide a new treatment option for unresectable metastatic melanoma in the liver.

Covered Stents and Coil Embolization for Treatment of Postpancreatectomy Arterial Hemorrhage.

PURPOSE: To evaluate the efficacy and clinical outcomes associated with stent-graft placement and coil embolization for postpancreatectomy arterial hemorrhage (PPAH). MATERIALS AND METHODS: Retrospective review of 38 stent-graft and/or embolization procedures in 28 patients (23 men; mean age, 65.1 y) for PPAH between 2007 and 2014 was performed. Time of bleeding, source of hemorrhage, intervention and devices used, repeat intervention rate, time to recurrent bleeding, complications, and 30-day mortality were assessed. Independent risk factors for recurrent bleeding and 30-day mortality were identified. RESULTS: Median onset of hemorrhage was at 39 days (mean, 27.9 d; range, 5-182 d). Covered stents were used in 65.7% of interventions, coil embolization in 23.6%, stent-assisted embolization in 5.2%, and stent-graft angioplasty in 2.6%. A total of 28 stent-grafts were placed, of which 19 were self-expandable and nine were balloon-mounted. Mean stent-graft diameter was 6.6 mm (range, 5-10 mm). Recurrent bleeding occurred following 26.3% of interventions in seven patients at a mean interval of 22 days. The site of recurrent bleeding was new in 80% of cases. There was no significant difference in recurrent bleeding rate in early-onset (< 30 d; n = 22) versus late-onset PPAH (> 30 d; n = 6; P > .05). No ischemic hepatic or bowel complications were identified. The 30-day mortality rate was 7.1% (n = 2) and was significantly higher in patients with initial PPAH at ≥ 39 days (n = 5; P = .007). CONCLUSIONS: Covered stents and coil embolization are effective for managing PPAH and maintaining distal organ perfusion to minimize morbidity and mortality. Recurrent bleeding is common and most often occurs from new sites of vascular injury rather than previously treated ones.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

The United States Culture Collection Network (USCCN): Enhancing Microbial Genomics Research through Living Microbe Culture Collections.

The mission of the United States Culture Collection Network (USCCN; http://usccn.org) is "to facilitate the safe and responsible utilization of microbial resources for research, education, industry, medicine, and agriculture for the betterment of human kind." Microbial culture collections are a key component of life science research, biotechnology, and emerging global biobased economies. Representatives and users of several microbial culture collections from the United States and Europe gathered at the University of California, Davis, to discuss how collections of microorganisms can better serve users and stakeholders and to showcase existing resources available in public culture collections.

Neurospora crassa: looking back and looking forward at a model microbe.

Investigation of the red bread mold that contaminated French bakeries nearly two centuries ago has led to a wealth of discoveries that have impacted our understanding of genetic, biochemical, and molecular mechanisms in microbes, from Mendelian genetics and the gene-enzyme relationship to circadian rhythm and plant cell wall degradation. Early Neurospora research focused on elucidating mechanisms of genetic recombination and gene action and later progressed to addressing complex biological questions of eukaryotic microbes. Here we review the evolution of the filamentous fungus Neurospora as a model microbe over the past century. We discuss the origins of Neurospora as a model microbe, the immediate scientific impacts from work in this filamentous fungus, and how the introduction of other model organisms (i.e., Escherichia coli and Saccharomyces cerevisiae) redirected the focus of Neurospora research. Neurospora has and continues to inform our understanding of a myriad of basic scientific concepts and now has the opportunity to forge into the applied biosciences and biotechnology.

Combined interventional radiology followed by endoscopic therapy as a single procedure for patients with failed initial endoscopic biliary access.

BACKGROUND: Percutaneous transhepatic cholangiography (PTC) assisted endoscopic retrograde cholangiopancreatography (ERCP) usually requires two separate sessions. There are no reports to support performing the procedures in a single session. AIM: The purpose of this study was to assess the feasibility and safety of the ERCP rendezvous technique via PTC in a single session for patients with initially failed endoscopic biliary intervention. METHOD: We conducted a retrospective cohort study in a high volume tertiary referral center. A single experienced endoscopist and two interventional radiologists performed all the procedures. Patient demographics and all the related clinical data from January 2009 to July 2011 were obtained from hospital records. Outcome measures were the overall success rates of completion of the combined PTC and ERCP sessions for biliary drainage. Procedure-related complications (bleeding, perforation, hemobilia, bile leak, pancreatitis or cholangitis) were also assessed. RESULT: Twenty-three patients (14 men) with a median age of 68 years (range 47-89 years) underwent 26 combined PTC-ERCP as a single procedure. PTC and ERCP were both performed within 6 h of failed ERCP in 19 cases (73 %) and the others within 72 h. A total of 91 % of patients had underlying gastrointestinal metastatic cancers, and a surgically altered pancreaticobiliary system was found in 26 % of patients. Percutaneous biliary access was obtained via PTC in all procedures and successful rendezvous therapy was performed in 23 cases (88 %), which include biliary stone removal with a balloon catheter (n = 7) and biliary prostheses (n = 19). The median procedure length for successful PTC-ERCP rendezvous was 60 min (range 14-147 min). With the mean follow-up of 202 days (range 8-833 days), three immediate procedural complications [asymptomatic pneumoperitoneum (n = 2) and post biliary sphincterotomy bleeding (n = 1)] and two delayed complications (a hemorrhagic shock from a damaged branch of hepatic artery and a biloma with secondary infection) occurred, and there was no procedure-associated mortality. CONCLUSION: This is the first report assessing the feasibility and safety of a combined procedure of ERCP and PTC in a single session. In experienced hands, the combined approach in a single session is appropriate in selected patients with an acceptable risk.

Global catalogue of microorganisms (gcm): a comprehensive database and information retrieval, analysis, and visualization system for microbial resources.

BACKGROUND: Throughout the long history of industrial and academic research, many microbes have been isolated, characterized and preserved (whenever possible) in culture collections. With the steady accumulation in observational data of biodiversity as well as microbial sequencing data, bio-resource centers have to function as data and information repositories to serve academia, industry, and regulators on behalf of and for the general public. Hence, the World Data Centre for Microorganisms (WDCM) started to take its responsibility for constructing an effective information environment that would promote and sustain microbial research data activities, and bridge the gaps currently present within and outside the microbiology communities. DESCRIPTION: Strain catalogue information was collected from collections by online submission. We developed tools for automatic extraction of strain numbers and species names from various sources, including Genbank, Pubmed, and SwissProt. These new tools connect strain catalogue information with the corresponding nucleotide and protein sequences, as well as to genome sequence and references citing a particular strain. All information has been processed and compiled in order to create a comprehensive database of microbial resources, and was named Global Catalogue of Microorganisms (GCM). The current version of GCM contains information of over 273,933 strains, which includes 43,436 bacterial, fungal and archaea species from 52 collections in 25 countries and regions.A number of online analysis and statistical tools have been integrated, together with advanced search functions, which should greatly facilitate the exploration of the content of GCM. CONCLUSION: A comprehensive dynamic database of microbial resources has been created, which unveils the resources preserved in culture collections especially for those whose informatics infrastructures are still under development, which should foster cumulative research, facilitating the activities of microbiologists world-wide, who work in both public and industrial research centres. This database is available from http://gcm.wfcc.info.

An uncommon cause of gross hematuria: two cases of ureteroarterial fistula.

Ureteroarterial fistula (UAF) is a rare but potentially fatal cause of hematuria seen in patients with prolonged ureteral stenting in the setting of surgery or abdominal radiation. It presents as episodic, transfusion-dependent hematuria with formation of clots. There is no current consensus for the optimal way to diagnose or treat UAF. We report two cases of UAF that required repeated studies and provocative testing to confirm the diagnosis. Both were successfully managed by endovascular stenting. Clinicians must recognize the diagnostic difficulties involved and maintain a high index of suspicion for UAF in stented patients with intermittent, severe hematuria.

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.

Best practices for fungal germplasm repositories and perspectives on their implementation.

In over 50 years, the Fungal Genetics Stock Center has grown to become a world-recognized biological resource center. Along with this growth comes the development and implementation of myriad practices for the management and curation of a diverse collection of filamentous fungi, yeast, and molecular genetic tools for working with the fungi. These practices include techniques for the testing, manipulation, and preservation of individual fungal isolates as well as for processing of thousands of isolates in parallel. In addition to providing accurate record keeping, an electronic managements system allows the observation of trends in strain distribution and in sample characteristics. Because many ex situ fungal germplasm repositories around the world share similar objectives, best-practice guidelines have been developed by a number of organizations such as the Organization for Economic Cooperation and Development or the International Society for Biological and Environmental Repositories. These best-practice guidelines provide a framework for the successful operation of collections and promote the development and interactions of biological resource centers around the world.

Identifying the gluc-1 and gluc-2 mutations in Neurospora crassa by genome resequencing.

Genome resequencing is an efficient strategy for associating mutant phenotypes with physical genomic loci (Baker 2009). A pilot study of this approach demonstrated that the Neurospora crassa genetic map was critical in narrowing the possible candidate mutations in a strain to a small number in a limited, defined region of the genome (McCluskey et al. 2011). In this study, we utilize a resequencing strategy to identify the mutations underlying the gluc-1 and gluc-2 genes in N. crassa.

Phenotype to genotype in Neurospora crassa: Association of the scumbo phenotype with mutations in the gene encoding ceramide C9-methyltransferase.

Using a legacy of genetic mutants of Neurospora crassa, paired with resequencing efforts through JGI, we have identified the gene responsible for the 'scumbo' mutant. This early morphological mutant was described as "Irregular flat, spreading growth with knobby protrusions and abnormal conidiation, but no free conidia. Mycelium usually appears yellowish rather than orange. Female fertile." (Perkins, Radford et al. 2000). Our further investigation has found new insights into the identity and associated functions of scumbo as a ceramide C9 methyltransferase, previously annotated as "similar to cyclopropane-fatty-acyl-phospholipidsynthase", encoded by the gene NCU07859. This enzyme performs a fungal-specific methyl modification of glycosyl-ceramides and has implications for membrane homeostasis and hyphal polarity in filamentous fungi.

Trans-hepatic trans-portal selective angioembolization; a management option for severe acute variceal bleeding of ileal conduit stoma.

We present a case of severe acute variceal bleeding in an ileal conduit stoma successfully managed with trans-hepatic trans-portal selective angioembolization as a lifesaving measure. Despite repeated transfusions, the patient's hemoglobin continued to be unstable. The patient underwent transhepatic embolization of ileal stoma varicose veins. Angioembolization was followed up with excision of ileal conduit stoma and creation of cutaneous ureterostomy for definitive treatment management of hemorrhage. In conclusion, trans-hepatic trans-portal embolization is an effective option for management of severe acute variceal bleeding in an ileal conduit stoma as a lifesaving measure and can be followed by excision of the conduit.