The Altemeier procedure using biologic mesh.

Transperineal rectosigmoidectomy is a relatively safe operation for the treatment of rectal prolapse; however, it carries a high rate of recurrence. The use of biologic mesh to buttress the repair and support the pelvic floor muscles may decrease the rate of recurrence. The technique for performing an Altemeier procedure with biologic mesh is described.

Robotic TransAnal Minimally Invasive Surgery in a cadaveric model.

The technique of TransAnal Minimally Invasive Surgery (TAMIS) was pioneered in 2009 as a hybrid approach to endoluminal resections of appropriately selected rectal lesions. There are, however, limitations to performing this type of resection. Robotic TAMIS is a novel, experimental technique and in this study was performed in a cadaveric model at a surgical education center. Various tasks were carried out using robotic TAMIS, including full-thickness sharp and cautery excision of rectal wall, as well as intra-luminal suturing of the surgical defect. It was found that for the da Vinci-trained surgeon, these tasks were simple to perform and accomplished with greater precision when compared to standard TAMIS. Our initial results indicate that robotic TAMIS overcomes the limitations of standard TAMIS and that it is a feasible platform for transanal surgery. The cost, however, of performing robotic TAMIS may limit its application to special cases in which standard TAMIS or transanal endoscopic microsurgery resections may prove difficult. Further study is necessary to validate these preliminary findings before robotic TAMIS is performed on live patients.

Asexual sporulation in Aspergillus nidulans.

The formation of mitotically derived spores, called conidia, is a common reproductive mode in filamentous fungi, particularly among the large fungal class Ascomycetes. Asexual sporulation strategies are nearly as varied as fungal species; however, the formation of conidiophores, specialized multicellular reproductive structures, by the filamentous fungus Aspergillus nidulans has emerged as the leading model for understanding the mechanisms that control fungal sporulation. Initiation of A. nidulans conidiophore formation can occur either as a programmed event in the life cycle in response to intrinsic signals or to environmental stresses such as nutrient deprivation. In either case, a development-specific set of transcription factors is activated and these control the expression of each other as well as genes required for conidiophore morphogenesis. Recent progress has identified many of the earliest-acting genes needed for initiating conidiophore development and shown that there are at least two antagonistic signaling pathways that control this process. One pathway is modulated by a heterotrimeric G protein that when activated stimulates growth and represses both asexual and sexual sporulation as well as production of the toxic secondary metabolite, sterigmatocystin. The second pathway apparently requires an extracellular signal to induce sporulation-specific events and to direct the inactivation of the first pathway, removing developmental repression. A working model is presented in which the regulatory interactions between these two pathways during the fungal life cycle determine whether cells grow or develop.

Upstream elements repress premature expression of an Aspergillus developmental regulatory gene.

The Aspergillus nidulans abaA gene regulates intermediate steps in asexual reproductive development and is itself developmentally regulated. An 822-base-pair DNA fragment from the abaA 5'-flanking region is sufficient to drive developmentally appropriate expression of the Escherichia coli lacZ gene. Deletion analysis showed that this fragment contains elements that repress transcription in vegetative cells and immature conidiophores and that activate transcription later during development. A 45-base-pair region encompassing the major and minor abaA transcription initiation sites contains directly repeated sequences related to the mammalian initiator (Inr) element (S. T. Smale and D. Baltimore, Cell 57:103-113, 1989). This element or sequences in the untranslated leader were sufficient for correct transcription initiation and for measurable developmental induction. Similar elements were present at or near the initiation sites of other developmentally regulated genes. We propose that the temporal and spatial specificity of expression of these genes results from modulation of the activity of Inr elements.

brlA requires both zinc fingers to induce development.

Expression of the Aspergillus nidulans brlA gene induces a developmental pathway leading to the production of asexual spores. We have introduced mutations into brlA that are expected to disrupt either or both Cys2-His2 Zn(II) coordination sites postulated to exist in the BrlA polypeptide. The resultant brlA alleles fail to induce either the asexual reproductive pathway or the expression of development-specific genes. These data support the hypothesis that brlA encodes a nucleic acid-binding protein whose activity requires each of two zinc fingers.

Isolation of a gene required for programmed initiation of development by Aspergillus nidulans.

In contrast to many other cases in microbial development, Aspergillus nidulans conidiophore production initiates primarily as a programmed part of the life cycle rather than as a response to nutrient deprivation. Mutations in the acoD locus result in "fluffy" colonies that appear to grow faster than the wild type and proliferate as undifferentiated masses of vegetative cells. We show that unlike wild-type strains, acoD deletion mutants are unable to make conidiophores under optimal growth conditions but can be induced to conidiate when growth is nutritionally limited. The requirement for acoD in conidiophore development occurs prior to activation of brlA, a primary regulator of development. The acoD transcript is present both in vegetative hyphae prior to developmental induction and in developing cultures. However, the effects of acoD mutations are detectable only after developmental induction. We propose that acoD activity is primarily controlled at the posttranscriptional level and that it is required to direct developmentally specific changes that bring about growth inhibition and activation of brlA expression to result in conidiophore development.

Coordinate control of secondary metabolite production and asexual sporulation in Aspergillus nidulans.

Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.

Lipid characteristics of subcutaneous adipose tissue and M. longissimus thoracis of Angus and Wagyu steers fed to US and Japanese endpoints.

We hypothesized that the concentrations of monounsaturated fatty acids (MUFA) and cholesterol of adipose tissue and M. longissimus thoracis would not differ between Angus and American Wagyu steers when fed to a typical US live weight, but would diverge when fed to a Japanese live weight. To test this, 8 steers of each breed type were assigned to a high-energy, corn-based diet, and another 8 steers of each breed type were fed coastal bermuda grass hay diet, supplemented with the corn-based diet to achieve a daily gain of 0.9kg/d. Targeted final body weights were 525kg for steers fed for 8 or 12mo the corn- or hay-based diets, respectively, and were 650kg for steers fed for 16 or 20mo the corn- or hay-based diets. Digesta concentrations of stearic (18:0) and trans-vaccenic acid decreased, whereas linoleic acid (18:2n-6) increased between the US and Japanese endpoints (all P⩽0.03). α-Linolenic acid (18:3n-3) increased in digesta only in the hay-fed steers during this time. Plasma concentrations of palmitic (16:0) and palmitoleic acid (16:1n-7), and the 16:1:18:0 ratio, were higher in Angus steers than in Wagyu steers. Also, the plasma 16:1:18:0 ratio was decreased by hay feeding in Angus steers, but increased in Wagyu steers, when fed to the Japanese endpoint. Concentrations of oleic (18:1n-9), linoleic, α-linolenic, and 18:2trans-10,cis-12 conjugated linoleic acid all were higher in Wagyu than in Angus subcutaneous (s.c.) adipose tissue, whereas myristic (14:0) and palmitic acid were higher in Angus s.c. adipose tissue (P⩽0.05). All MUFA increased, and saturated fatty acids decreased, between the US and Japanese endpoints. Slip points of lipids in s.c. adipose tissue were over 10°C lower (P=0.01) in Japanese-endpoint steers than in US endpoint steers, consistent with the overall increase in MUFA with time on feed. The concentration of cholesterol in the M. longissimus thoracis increased with time, which may have been related to the increase in oleic acid. Because the breed×endpoint interaction was not significant for cholesterol or any of the adipose tissue fatty acids, we conclude that our original hypothesis was incorrect. Of the three factors tested (breed type, diet, and slaughter age endpoint), endpoint had the greatest effect on adipose tissue lipid composition.

Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse-human tumor models.

Monoclonal antibodies (MoAbs) against carcinoembryonic antigen were successfully radiolabeled with 111In, and the radiopharmaceutical was characterized in vitro and in normal and tumor-bearing mice. The 111In-MoAb proved to be stable in vitro and in vivo under normal conditions, although instability could be induced in vitro with large quantities of iron-free transferrin. Animal distribution studies with 111In-MoAb demonstrated tumor localization superior to 67Ga and pharmacokinetics that were highly similar to those of endogenously labeled 75Se-MoAb. The 111In-MoAb followed first-order kinetics and fit a two-compartmental model when studied in nude mice bearing human colon tumors known to express carcinoembryonic antigen. Significant quantities of radiolabel appeared in tissues other than tumor, with liver and skin having the highest concentrations. Sufficient tumor/background ratios were formed for scanning purposes. The data indicate that 111In-MoAb may prove to be effective as a radiopharmaceutical for tumor imaging.

Identification of developmental regulatory genes in Aspergillus nidulans by overexpression.

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [alcA(p)] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA(p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA(p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA(p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA(p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA(p)::brlA induction. Sequence analyses of the DNA fragments under alcA(p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA(p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.

Aspergillus fabM encodes an essential product that is related to poly(A)-binding proteins and activates development when overexpressed.

Overexpression of several different Aspergillus nidulans developmental regulatory genes has been shown to cause inappropriate developmental activation and growth inhibition. We previously exploited this observation that induced development caused growth inhibition in designing a screen to identify other genes that could activate development when overexpressed. We identified 16 mutants in which induced expression of different random genomic DNA sequences caused growth inhibition, accumulation of mRNA corresponding to the brlA developmental regulatory locus, and in several cases sporulation. This phenotype was designated FAB for Forced expression Activation of brlA and the genes were called fabA through fabP. Here we describe one of these genes, fabM, which is predicted to encode a poly(A)-binding protein (PABP) that is constitutively expressed and is essential for viability. While it is unclear why overexpression of the fabM caused sporulation, we showed that this activity required other known early developmental regulators including brlA beta, flbA, flbB, flbC, and fluG. We propose that fabM is an example of a gene that is not only required for growth, but also has specific functions early in development that assist, developmental induction, presumably by allowing translation of specific mRNAs like brlA.

Extragenic suppressors of loss-of-function mutations in the aspergillus FlbA regulator of G-protein signaling domain protein.

We showed previously that two genes, fl bA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the alpha subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. fl bA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a fl bA loss-of-function mutation ( fl bA98). These suppressors overcome fl bA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild type whereas sfaA1 is semidominant. sfaA1 also differs from other suppressor mutations in that it cannot suppress a fl bA deletion mutation (and is therefore allele specific) whereas all the dominant suppressors can bypass complete loss of fl bA. Only sfaE83 suppressed dominant activating mutations in fadA, indicating that sfaE may have a unique role in fadA- fl bA interactions. Finally, none of these suppressor mutations bypassed fl uG loss-of-function mutations in development-specific activation.

A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans.

We investigated the functions of the highly expressed, sporulation-specific SpoC1 genes of Aspergillus nidulans by deleting the entire 38-kb SpoC1 gene cluster. The resultant mutant strain did not differ from the wild type in (1) growth rate, (2) morphology of specialized reproductive structures formed during completion of the asexual or sexual life cycles, (3) sporulation efficiency, (4) spore viability or (5) spore resistance to environmental stress. Thus, deletion of the SpoC1 gene cluster, representing 0.15% of the A. nidulans genome, had no readily detectable phenotypic effects. Implications of this result are discussed in the context of major alterations in gene expression that occur during A. nidulans development.

Aspergillus nidulans mutants defective in stc gene cluster regulation.

The genes involved in the biosynthesis of sterigmatocystin (ST), a toxic secondary metabolite produced by Aspergillus nidulans and an aflatoxin (AF) precursor in other Aspergillus spp., are clustered on chromosome IV of A. nidulans. The sterigmatocystin gene cluster (stc gene cluster) is regulated by the pathway-specific transcription factor aflR. The function of aflR appears to be conserved between ST- and AF-producing aspergilli, as are most of the other genes in the cluster. We describe a novel screen for detecting mutants defective in stc gene cluster activity by use of a genetic block early in the ST biosynthetic pathway that results in the accumulation of the first stable intermediate, norsolorinic acid (NOR), an orange-colored compound visible with the unaided eye. We have mutagenized this NOR-accumulating strain and have isolated 176 Nor(-) mutants, 83 of which appear to be wild type in growth and development. Sixty of these 83 mutations are linked to the stc gene cluster and are likely defects in aflR or known stc biosynthetic genes. Of the 23 mutations not linked to the stc gene cluster, 3 prevent accumulation of NOR due to the loss of aflR expression.

Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans.

We showed previously that a DeltafluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG ( approximately 400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of DeltafluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from DeltaflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused conidiation in submerged A. nidulans cultures just as was previously observed for overexpression of flbD. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD and this is the first demonstration of functional complementation of an A. nidulans sporulation defect using a gene from an evolutionarily distant fungus. However, deletion of the rca-1 gene in N. crassa had no major effect on growth rate, macroconidiation, microconidiation, or ascospore formation. The only phenotype displayed by the rca-1 mutant was straight or counterclockwise hyphal growth rather than the clockwise spiral growth observed for wild type. Thus, if rca-1 is involved in N. crassa development, its role is subtle or redundant.

Bidirectional gene transfer between Aspergillus fumigatus and Aspergillus nidulans.

A genomic DNA library was constructed from a pathogenic strain of Aspergillus fumigatus using the cosmid vector pCosAX. Cosmid clones with homologies to the rodA, brlA, fluG, flbA or trpC genes from A. nidulans were isolated from the library. Each A. fumigatus clone was used to complement a strain of A. nidulans with a mutation in the homologous gene. A spontaneous white spored strain of A. fumigatus was isolated. The mutation was complemented by transforming the strain with a plasmid containing the wA gene from A. nidulans. The results from these experiments indicate a significant degree of structural and functional homology between genes from the organisms. These findings indicate the potential to exploit the methods and information available from A. nidulans to address questions related to human disease caused by A. fumigatus and the ability to use A. nidulans as a surrogate genetic host for characterizing A. fumigatus gene function.

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway.

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.