Management of neuromuscular diseases and spinal muscular atrophy in Latin America.

Latin America (LA) has a population of ~645 million people distributed over 33 countries with marked political, cultural and economic differences. In LA, patients with inherited neuromuscular diseases (NMDs) often do not have access to specialized medical centers and many of them go undiagnosed. General management and care of spinal muscular dystrophy (SMA) patients in the region varies due to heterogeneous health care. An active generation of young clinical neurologists is being trained for the specialized care of SMA and other neuromuscular (NM) patients, both in the private and public sectors. The Euro-Latin-American Summer School of Myology (EVELAM) as well as efforts of professionals at large public centers in the major cities of LA have a leading role in this development. Different regional academic-scientific organizations as well as the expanding number of telethon centers and the creation of parent organizations, mostly concerning SMA, all together are contributing to the increased quality of the management of NMD patients. Over the past years, academic and clinical research, as well as the establishment of qualified centers for the molecular testing of NMD are pushing forward the creation of patient registries and the development of specific clinical trials, with Argentina and Brazil having a major role in this field. Nevertheless, increased awareness and further training of specialized health professionals are necessary to reach patients that are currently lacking care throughout the region.

Osteogenic cell response to calcium aluminate-based cement.

AIM: To evaluate the effect of a calcium aluminate-based cement (CAC+) on the development of the osteogenic phenotype in vitro. METHODOLOGY: Rat calvaria-derived cells were grown on Thermanox® coverslips for 24 h and then exposed to either samples (4-h set) of CAC+ or mineral trioxide aggregate (MTA) placed on Transwell® inserts for periods of up to 14 days. Nonexposed cultures were used as the controls. The comparisons were made using the nonparametric Kruskal-Wallis test, followed by the Student-Newman-Keuls post hoc test when appropriate. RESULTS: The results showed that proximity to MTA or CAC+ samples inhibited cell growth, whereas at a distance, viable and proliferative cells adhered to and spread on the Thermanox® , expressing osteoblast differentiation markers prior to mineralization of the extracellular matrix. Compared with MTA, the osteogenic cell cultures exposed to CAC+ exhibited significantly greater cell viability, alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2, osterix, ALP, bone sialoprotein and osteocalcin (P < 0.05 for all). For the osteogenic cell cultures exposed to CAC+, the quantification of matrix mineralization was not altered (P > 0.05). CONCLUSIONS: CAC+ supported the acquisition of the osteogenic cell phenotype in vitro, rendering this novel material a potential alternative to MTA in endodontic procedures. Further in vivo studies are needed to verify if the beneficial in vitro effects of CAC+ on osteoblastic cells correspond to an increase and/or acceleration of bone repair in the periapical region.

[Thoracic inestability fixed with bioabsorbable screws and plates]. / Fijación de tórax inestable con placas y tornillos bioabsorbibles. Presentación de serie de casos.

BACKGROUND: Flail chest is managed with mechanical ventilation or inhaloteraphy and analgesia. Little has been published on the use of bioabsorbable material and its evolution in flail chest fixation. METHODS: This is a descriptive study of patients with unstable chest undergoing fixation with bioabsorbable plates and screws in a period from February 2009 to December 2011. RESULTS: We report 18 cases, aged 33-74 years (mean 53), three with bilateral involvement; rib fixation was performed between 1-21 days of the accident (mean 1.5). They started walking the next day in 14 cases; postoperative stay was four days (range 3-14). The heart rate of patients prior to surgery was 90 per minute (range 82-100) and lowered to 84 after fixation (range 82-92), preoperative respiratory rate was 26 per minute (range 22-28) and below 22 per minute (range 20 to 26) in postoperative period. CONCLUSIONS: The use of bioabsorbable material for osteosynthesis of costal fractures did not show side effects in our period of study.

ANTECEDENTES: El tórax inestable se trata con ventilación mecánica o inhaloterapia y analgesia. Poco se ha publicado sobre el uso de material bioabsorbible y su evolución en la fijación de tórax inestable. MÉTODOS: Estudio descriptivo de pacientes con tórax inestable sometidos a fijación con placas y tornillos bioabsorbibles en un período comprendido de Febrero de 2009 a Diciembre de 2011. RESULTADOS: Presentamos 18 casos con edades entre 33 y 74 años (media de 53), tres con tórax inestable bilateral; la fijación costal se realizó entre 1 y 21 días del accidente (media de 1.5). Se inició deambulación al día siguiente en 14 casos; la estancia postoperatoria fue de cuatro días (rango de 3 a 14). La frecuencia cardíaca de los pacientes previa a la cirugía era de 90 por minuto (rango 82 a 100) y bajó a 84 después de la fijación (rango 82 a 92); la frecuencia respiratoria preoperatoria era 26 por minuto (rango 22 a 28) y bajó a 22 por minuto (rango 20 a 26) en el postoperatorio. CONCLUSIONES: El uso de material bioabsorbible para osteosíntesis costal no tiene efectos secundarios atribuibles al material en el corto plazo.

Effect of autogenous and fresh-frozen bone grafts on osteoblast differentiation.

OBJECTIVE: Fresh-frozen bone allograft (FFBA) is an alternative to autogenous bone (AB) for reconstructing maxillary bone. Despite the promising clinical results, cell responses to FFBA and AB were not evaluated. Thus, our aim was to compare cells harvested from maxillary reconstructed sites with either AB or FFBA in terms of osteoblast differentiation and to evaluate the effect of culturing cells in contact with FFBA. METHODS: Cells harvested from three patients submitted to bilateral maxillary reconstruction with AB and FFBA were cultured to evaluate: proliferation, alkaline phosphatase activity, extracellular matrix mineralization and gene expression of osteoblastic markers. The effect of FFBA on osteoblast differentiation was studied by culturing cells harvested from AB in contact with FFBA and evaluating the same parameters. Data were compared using either two-way ANOVA followed by Tukey-b test or Student's t test (p≤0.05). RESULTS: Cell proliferation was higher in cultures from AB grafted sites and extracellular matrix mineralization was higher in cultures derived from FFBA grafted sites. The gene expression of alkaline phosphatase, RUNX2, bone sialoprotein and osteocalcin was higher in cells derived from FFBA compared with cells from AB grafted sites. However, the exposure of cells derived from AB to FFBA particles did not have any remarkable effect on osteoblast differentiation. CONCLUSIONS: These results indicate the higher osteogenic activity of cells derived from FFBA compared with AB reconstructed sites, offering an explanation at cellular level of why FFBA could be a suitable alternative to AB for reconstructing maxillary bone defects.

Phthalocyanine adsorption to graphene on Ir(111): evidence for decoupling from vibrational spectroscopy.

Phthalocyanine molecules have been adsorbed to Ir(111) and to graphene on Ir(111). From a comparison of scanning tunneling microscopy images of individual molecules adsorbed to the different surfaces alone it is difficult to discern potential differences in the molecular adsorption geometry. In contrast, vibrational spectroscopy using inelastic electron scattering unequivocally hints at strong molecule deformations on Ir(111) and at a planar adsorption geometry on graphene. The spectroscopic evidence for the different adsorption configurations is supported by density functional calculations.

Nanotopography drives stem cell fate toward osteoblast differentiation through α1ß1 integrin signaling pathway.

The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2 SO4 /H2 O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non-osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non-osteogenic conditions. Additionally, the gene expression of α1 and ß1 integrins was higher in cells grown on Ti with nanotopography under non-osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1ß1 integrin inhibitor. These results indicate that α1ß1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface-mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration.

Comparative study of bone repair in mandibular body osteotomy between metallic and absorbable 2.0 mm internal fixation systems. Histological and histometric analysis in dogs: a pilot study.

The objective of this study was to compare the bone repair along a mandibular body osteotomy stabilized with 2.0 mm absorbable and metallic systems. 12 male, adult mongrel dogs were divided into two groups (metallic and absorbable) and subjected to unilateral osteotomy between the mandibular third and fourth premolars, which was stabilized by applying two 4-hole plates. At 2 and 18 weeks, three dogs from each group were killed and the osteotomy sites were removed and divided equally into three parts: the upper part was labelled the tension third (TT), the lower part the compression third (CT), and the part between the TT and CT the intermediary third (IT). Regardless of the treatment system, union between the fragments was observed at 18 weeks and the CT showed more advanced stages of bone repair than the TT. Histometric analysis did not reveal any significant differences among the 3 parts or systems in the distance between bone fragments at 2 weeks. Although at 18 weeks the proportions of newly formed bone did not differ among TT, IT and CT, significantly enhanced bone formation was observed in all sections for the metallic group. The patterns of repair were distinct between treatments.

The influence of pore size on osteoblast phenotype expression in cultures grown on porous titanium.

This study investigated the effect of pore size on osteoblastic phenotype development in cultures grown on porous titanium (Ti). Porous Ti discs with three different pore sizes, 312 µm (Ti 312), 130 µm (Ti 130) and 62 µm (Ti 62) were fabricated using a powder metallurgy process. Osteoblastic cells obtained from human alveolar bone were cultured on porous Ti samples for periods of up to 14 days. Cell proliferation was affected by pore size at day 3 (p=0.0010), day 7 (p=0.0005) and day 10 (p=0.0090) in the following way: Ti 62

A complete set of self-consistent charge density-functional tight-binding parametrization of zinc chalcogenides (ZnX; X=O, S, Se, and Te).

We have developed a complete set of self-consistent charge density-functional tight-binding parameters for ZnX (X = Zn, O, S, Se, Te, Cd, H, C, and N). The transferability of the derived parameters has been tested against Pseudo Potential-Perdew, Burke and Ernzerhof (PP-PBE) calculations and experimental values (whenever available) for corresponding bulk systems (e.g., hexagonal close packing, zinc-blende, and wurtzite(wz)), various kinds of nanostructures (such as nanowires, surfaces, and nanoclusters), and also some small molecular systems. Our results show that the derived parameters reproduce the structural and energetic properties of the above-mentioned systems very well. With the derived parameter set, one can study zinc-chalcogenide nanostructures of relatively large size which was otherwise prohibited by other methods. The Zn-Cd parametrization developed in this article will help in studying large semiconductor hetero-nanostructures of Zn and Cd chalcogenides such as ZnX/CdX core/shell nanoparticles, nanotubes, nanowires, and nanoalloys.

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells.

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non-osteogenic medium with or without purmorphamine (2 µM) for periods of up to 14 days. Purmorphamine up-regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage.

Self-Consistent-Charge Density-Functional Tight-Binding Parameters for Cd-X (X = S, Se, Te) Compounds and Their Interaction with H, O, C, and N.

Parameters for CdX, SeX, and TeX (X = H, C, N, O, S, Se, Te, and Cd) have been generated within the self-consistent-charge density-functional tight-binding (SCC-DFTB) framework. The approach has been tested against ab initio density-functional theory calculations for the relevant bulk phases, surfaces, nanowires, and small molecular systems. The SCC-DFTB approach reproduces structural, electronic, and energetic properties very well, demonstrating that the developed parameters are fully transferable among different chemical environments.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane.

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron- and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Clinical features of facioscapulohumeral muscular dystrophy 2.

OBJECTIVE: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss of DNA methylation and heterochromatin markers at the D4Z4 repeat that are similar to patients with D4Z4 contractions (FSHD1). This commonality suggests that a change in D4Z4 chromatin structure unifies FSHD1 and FSHD2. The aim of our study was to critically evaluate the clinical features in patients with FSHD2 in order to establish whether these patients are phenotypically identical to FSHD1 and to establish the effects of the (epi-) genotype on the phenotype. METHODS: This cross-sectional study studied 33 patients with FSHD2 from 27 families, the largest cohort described to date. All patients were clinically assessed using a standardized clinical evaluation form. Genotype analysis was performed by pulsed field gel electrophoresis and PCR; D4Z4 methylation was studied by methylation-sensitive Southern blot analysis. RESULTS: FSHD2 is identical to FSHD1 in its clinical presentation. Notable differences include a higher incidence (67%) of sporadic cases and the absence of gender differences in disease severity in FSHD2. Overall, average disease severity in FSHD2 was similar to that reported in FSHD1 and was not influenced by D4Z4 repeat size. In FSHD2, a small effect of the degree of hypomethylation on disease severity was observed. CONCLUSIONS: Clinically, patients with FSHD2 are indistinguishable from patients with FSHD1. The present data suggest that FSHD1 and FSHD2 are the result of the same pathophysiologic process.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes.

The aim of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR). Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20,000 cells well(-1) and cultured for up to 21 days. Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF. Using a higher cell density, real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1, 10 and 14. Expression of the apoptotic genes bax, bcl-2 and survivin was evaluated for both cultures. hPDLF adhered and spread more on P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater on P(VDF-TrFE)/BT at 2 and 4h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7, no signs of keratinocyte proliferation could be noticed for both membranes. Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed on P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion, spreading, proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR.

Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease.

INTRODUCTION: Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-beta (TGF-beta), IL-1beta, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. METHODS: Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-beta, IL-1beta, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-kappaB ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-beta, IL-1beta, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. RESULTS: Our data demonstrated elevated levels of IL-17, TGF-beta, IL-1beta, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. CONCLUSION: These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease.

Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases.

INTRODUCTION: Periapical chronic lesion formation involves activation of the immune response and alveolar bone resorption around the tooth apex. However, the overall roles of T helper type 1 (Th1), Th2, and T-regulatory cell (Treg) responses and osteoclast regulatory factors in periapical cysts and granulomas have not been fully determined. This study aimed to investigate whether different forms of apical periodontitis, namely cysts and granulomas, show different balances of Th1, Th2 regulators, Treg markers, and factors involved in osteoclast chemotaxis and activation. METHODS: Gene expression of these factors was assessed using quantitative real-time polymerase chain reaction, in samples obtained from healthy gingiva (n = 8), periapical granulomas (n = 20), and cysts (n = 10). RESULTS: Periapical cysts exhibited a greater expression of GATA-3, while a greater expression of T-bet, Foxp3, and interleukin-10 (IL-10) was seen in granulomas. The expression of interferon-gamma, IL-4, and transforming growth factor-beta was similar in both lesions. Regarding osteoclastic factors, while the expression of SDF-1alpha/CXCL12 and CCR1 was higher in cysts, the expression of RANKL was significantly higher in granulomas. Both lesions exhibited similar expression of CXCR4, CKbeta8/CCL23, and osteoprotegerin, which were significantly higher than in control. CONCLUSION: Our results showed a predominance of osteoclast activity in granulomas that was correlated with the Th1 response. The concomitant expression of Treg cell markers suggests a possible suppression of the Th1 response in granulomas. On the other hand, in cysts the Th2 activity is augmented. The mechanisms of periradicular lesion development are still not fully understood but the imbalance of immune and osteoclastic cell activity in cysts and granulomas seems to be critically regulated by Treg cells.

Resonant electron heating and molecular phonon cooling in single C60 junctions.

We study heating and heat dissipation of a single C(60) molecule in the junction of a scanning tunneling microscope by measuring the electron current required to thermally decompose the fullerene cage. The power for decomposition varies with electron energy and reflects the molecular resonance structure. When the scanning tunneling microscope tip contacts the fullerene the molecule can sustain much larger currents. Transport simulations explain these effects by molecular heating due to resonant electron-phonon coupling and molecular cooling by vibrational decay into the tip upon contact formation.

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization.

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al(2)O(3) particles 65 mum), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al(2)O(3) was detected by EDS and the amount calculated by digital analyses. Osteoblasts were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al(2)O(3) residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al(2)O(3) residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al(2)O(3) negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces.

Cytokine and chemokine response of bone cells after dentin challenge in vitro.

OBJECTIVE: The aim of this study was to characterize the effects of dentin extracts on cytokine, chemokine and nitric oxide (NO) production by primary rat bone cells. STUDY DESIGN: Osteoblastic bone marrow cultures were exposed to particulate (D-part), non-particulate (D-n-part) and demineralized dentin extracts and evaluated for proliferative activity, cell morphology, alkaline phosphatase activity and bone-like nodule formation. Cytokine production was assessed by enzyme-linked immunosorbent assay and NO release by the Griess method. RESULTS: The dentin extracts did not affect osteoblast numbering. Conversely, they up regulated in a dose-dependent manner the production by the osteoblasts of the pro-inflammatory interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, IL-6, cytokine-induced neutrophil chemoattractant-1, and of the anti-inflammatory cytokine, IL-10. The NO production was stimulated only by D-n-part. CONCLUSION: These results demonstrate that dentin induces the production of inflammatory cytokines by osteoblasts and suggest that pro-resorptive pathways might be stimulated when dentin molecules come into contact with bone cells during pathological processes associated with dentin and bone matrix dissolution.

The Neurospora crassa cfp promoter drives a carbon source-dependent expression of transgenes in filamentous fungi.

AIMS: The objective of the present study was to determine the potential of promoter sequences from the cfp gene of Neurospora crassa to drive the expression of transgenes in filamentous fungi. METHODS AND RESULTS: Northern blot analyses showed that the mRNA levels of cfp were rapidly modified in response to either inducing or repressing culture conditions. The hygromycin phosphotransferase (hph) and S-adenosylmethionine synthetase (eth-1) genes were fused to a minimal cfp promoter fragment (Pcfp) and used as reporter genes. These constructs were highly expressed in transformant N. crassa strains grown in media containing glucose or sucrose and repressed in media containing ethanol or ethanol plus glucose. A gene fusion of the cfp promoter to the beta-glucuronidase gene (cfp-uidA) showed identical patterns of expression in the heterologous filamentous fungus Aspergillus nidulans. CONCLUSIONS: Our results show that the levels of expression of the native cfp gene, as well as reporter genes driven by cfp promoter sequences, can be rapidly modified in response to different carbon sources. These modified levels of expression are maintained by continuous growth in the presence of the corresponding carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that the cfp promoter can be used to control the expression of transgenes in filamentous fungi in a carbon source-dependent fashion.