Intraosseous Venous Malformation of the Zygomatic Bone: Virtual Surgical Planning and Reconstruction With Customized CAD-CAM Titanium Implant.

ABSTRACT: Zygomatic intraosseous vascular anomalies are uncommon. Most cases have been reported without a precise diagnosis. The prevalence is 3:1 women during the fourth decade of life. The study of these lesions includes clinical findings, radiological studies, such as computed tomography and magnetic resonance imaging scans, and a postoperative histological study of the specimen. Biopsy is not recommended because of the risk of bleeding. Complete surgical resection remains the gold standard elective treatment.The case of a patient with an intraosseous venous malformation of the zygomatic bone is presented, detailing the clinical, histological, and radiological characteristics and the surgical treatment with a primary reconstruction through Computer-Aided Design/Computer-Aided Manufacturing customized titanium implant guided by virtual surgical planning with a reduction of surgical time and morbidity in the donor area with optimal functional and aesthetic results.

Secondary Cervicofacial Soft Tissue Reconstruction With Upper Trapezius Myocutaneous Flap in "Frozen Neck" With Bone Flap and Reconstructive Plate Exposure.

ABSTRACT: A frozen neck is a scarred neck with severe fibrosis with a loss of tissue planes secondary to prior irradiation with or without surgery. The purpose of this study was to evaluate the outcomes of cervicofacial reconstruction in patients with soft tissue defects and bone flap and reconstruction plate exposure with the upper trapezius myocutaneous flap. Fifteen oncologic patients with prior surgery and radiotherapy developed soft tissue dehiscence with bone and osteosynthesis material exposure. All patients had either a frozen neck or a vessel-depleted neck. The soft tissue defects were reconstructed, the osteosynthesis material was removed and the bone flap exposure was covered in all patients. One patient developed a seroma and 1 patient reported wound dehiscence. In terms of esthetic results, 6 patients referred a good esthetic result, whereas 8 patients referred a fair result and 1 patient a poor result. Two patients with prior radical neck dissection reported a poor functional result in the ipsilateral shoulder, previously to secondary reconstruction. Functional neck dissection was performed in 10 patients, 8 patients referred a good functional outcome and 2 patients reported a fair result. The upper trapezius flap is an extremely reliable source for secondary cervicofacial soft tissue reconstruction in "frozen neck." In comparison with other locoregional flaps, the upper trapezius flap fulfills all aesthetic and functional criteria for secondary cervicofacial soft tissue reconstruction.

Parascapular Flap for Severe Hidradenitis Suppurativa.

Hidradenitis suppurativa is a chronic inflammatory disease which affects apocrine glands and hair follicles of the skin, primarily in the axillary and groin regions. This condition can be highly debilitating, causing painful lesions and a negative psychological impact on patients. While medical and minimally invasive treatments are available, surgical intervention may be necessary for severe cases. In cases involving axillary defects, the use of local flaps such as the parascapular flap is a viable option. In this case report, we present a 34-year-old woman who presented to our clinic with a history of recurrent abscesses and cutaneous infections in the axillary region. After thorough evaluation, we chose to use the parascapular flap for reconstruction. The parascapular flap is a one-stage procedure that allows for extensive resection of the axillary area without resulting in contractions or retractions over the long term. Additionally, this technique allows for preservation of the axilla's original shape with minimal donor site morbidity.

Embryonal Rhabdomyosarcoma of the Tongue in Adults.

(1) Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the first two decades of life. One third of cases appear in the head and neck, with 60% of these being embryonal type. RMS is extremely rare in adults, comprising only 1% of adult malignancies, and of those, only 3.3% are rhabdomyosarcomas. (2) Case report: A 46 y.o. male presented with a 1 cm exophytic pediculated painless lesion on the dorsum of his tongue, with progressive growth for 3 months. An excisional biopsy revealed an "embryonal rhabdomyosarcoma with fusocellular areas, with negative rearrangement for gen FOXO1A, negative MDM2 (only focal positivity), and positive INI-1". Subsequent contrast-enhanced MRI concluded the presence of a lesion with imprecise margins in the right half-tongue, 15 × 8 × 7 mm (longitudinal × transverse × craniocaudal), compatible with a sarcoma. The patient underwent a partial centrolingual glossectomy followed by reconstruction with a buccinator muscle local flap. After surgery, he received chemotherapy with eight cycles of VAC (vincristine, actinomycin, and cyclophosphamide) protocol. The patient is now disease free after 42 months, with good tongue function. (3) Discussion and conclusions: Embryonal RMS is an extremely rare sarcoma in adults, and the location in the tongue is even more exceptional (only two more similar cases are reported in the literature). The prognosis in adults is significantly poorer than in children. A complete margin-free resection with an adequate chemotherapy protocol is the treatment of choice in cases such as these.

Depth of Invasion: Influence of the Latest TNM Classification on the Prognosis of Clinical Early Stages of Oral Tongue Squamous Cell Carcinoma and Its Association with Other Histological Risk Factors.

BACKGROUND: The American Joint Committee on Cancer (AJCC), in its 8th edition, introduces modifications to the previous TNM classification, incorporating tumour depth of invasion (DOI). The aim of this research is to analyse the prognosis (in terms of disease-free survival and overall survival) of clinical early stage (I and II) squamous cell carcinomas of the oral tongue according to the DOI levels established by the AJCC in its latest TNM classification to assess changes to the T category and global staging system and to evaluate the association between DOI and other histological risk factors. METHODS: A retrospective longitudinal observational study of a series of cases was designed. All patients were treated with upfront surgery at our institution between 2010 and 2019. The variables of interest were defined and classified into four groups: demographic, clinical, histological and evolutive control. Univariate and multivariate analyses were carried out and survival functions were calculated using the Kaplan-Meier method. Statistical significance was established for p values below 0.05. RESULTS: Sixty-one patients were included. The average follow-up time was 47.42 months. Fifteen patients presented a loco-regional relapse (24.59%) and five developed distant disease (8.19%). Twelve patients died (19.67%). Statistically significant differences were observed, with respect to disease-free survival (p = 0.043), but not with respect to overall survival (p = 0.139). A total of 49.1% of the sample upstaged their T category and 29.5% underwent modifications of their global stage. The analysis of the relationship between DOI with other histological variables showed a significant association with the presence of pathological cervical nodes (p = 0.012), perineural invasion (p = 0.004) and tumour differentiation grade (p = 0.034). Multivariate analysis showed association between depth of invasion and perineural invasion. CONCLUSIONS: Depth of invasion is a histological risk factor in early clinical stages of oral tongue squamous cell carcinoma. Depth of invasion impacts negatively on patient prognosis, is capable per se of modifying the T category and the global tumour staging, and is associated with the presence of cervical metastatic disease, perineural invasion and tumoural differentiation grade.

An Uncommon Great Pretender in Oral Cavity Lesions: The Masson's Tumor.

Intravascular papillary endothelial hyperplasia (IPEH) is a rare benign non-neoplastic vascular lesion. A typical presentation consists of a subcutaneous nodule that may simulate other clinical entities. Presentation in the oral cavity is uncommon. It is thought to develop as an abnormal proliferative reaction of endothelial cells in a process of impaired thrombogenesis. When endothelial proliferation occurs, a differential diagnosis with a soft tissue sarcoma, in particular an angiosarcoma, should be performed. We report a case of a 68-year-old female patient who presented with a lesion on the upper lip of 3 months' duration. Surgical resection revealed an IPEH. 1 year later, the patient showed a local recurrence requiring excision with clear margins. Pathological and immunohistochemical features can help us distinguish these lesions from those requiring more aggressive treatment. The gold standard is surgical resection with clear margins. Accurate preoperative diagnosis is essential to avoid overtreatment. Emphasis should be placed on clinical, radiological and histological studies.

Assessment of Quality of Life in Head-and-Neck Oncologic Patients with Intraoral Soft-Tissue Defects Reconstructed with Buccinator Myomucosal Flap.

The aim of this study is to evaluate the functional outcomes and quality of life (QoL) in oncologic patients with intraoral defects reconstructed with the buccinator myomucosal flap. A retrospective study was performed involving 39 patients with intraoral soft-tissue defects, reconstructed with a buccinator myomucosal flap during a six-year period. Patients completed the European Organization for Research and Treatment of Cancer questionnaires, the standard questionnaire (QLQ-C30) and the head-and-neck specific module (QLQ-H&N35). Thirty-nine patients with a mean age of 61.23 ± 15.80 years were included in the study. Thirty-three patients were diagnosed with an oncological condition (84.61%). Six patients (15.38%) developed orosinusal communication and underwent extensive debridement. The median global-health-status score was 79.27 and emotional performance was the lowest scoring, with a mean score of 76.93. As for the symptom items, the most outstanding were dental problems (33.33), oral opening (31.62) and dry mouth (37.61), followed by sticky saliva (24.79), problems with social eating (21.15) and pain (19.87). The most significant symptoms were radiotherapy-related adverse effects such as pain, fatigue, dental problems and dry mouth. Patients reconstructed with the buccinator myomucosal flap develop a good quality of life for all types of activities, and a correct function and aesthetics. Postoperative radiotherapy is associated with a poorer quality of life, and can lead to impairment of several symptoms such as swallowing, oral opening and dry mouth.

Functional Outcomes with Facial Artery Musculo-Mucosal (FAMM) Flap and Dental Implants for Reconstruction of Floor of the Mouth and Tongue Defects in Oncologic Patients.

Optimal functional outcomes in oncologic patients with squamous cell carcinoma (SCCA) of the tongue and floor of the mouth require good lingual mobility, adequate facial competence, the cheek suction effect and dental rehabilitation with osseointegrated implants. In this study, twenty-two oncologic patients who had been diagnosed with intraoral SCCA affecting the tongue and the floor of the mouth and who had undergone wide resection of the tumor and immediate reconstruction with an inferiorly pedicled FAMM flap and immediate osseointegrated implants were assessed. Lingual mobility, speech articulation, deglutition, implant success rate, mouth opening, and aesthetic results were evaluated. All patients were staged as T2 and the defect size ranged from 3.7 × 2.1 cm to 6.3 × 4.2 cm. A selective neck dissection was performed in all patients as part of their oncologic treatment, either electively or for node positive disease. Thirteen patients (59%) were diagnosed with node positive disease and underwent adjuvant radiotherapy. A total of 101 osseointegrated implants were placed for prosthetic rehabilitation and 8 implants were lost (7.9%), of which 7 received radiotherapy (87.5%). The implant success rate was 92.1%. Mouth opening was reported as normal in 19 patients (86.3%). Tongue tip elevation was reported as excellent in 19 patients (86.3%) and good in 3 patients (13.6%). Lingual protrusion was referred to as excellent in 15 patients (68.2%) and good in 6 patients (27.2%). Lateral excursion was reported as excellent in 14 patients (63.6%) and good in 7 patients (31.8%). In terms of speech articulation, 20 patients reported normal speech (90.9%). Regarding deglutition, 19 patients (86.3%) reported a regular diet while a soft diet was reported by 3 patients (13.7%). Aesthetic results were referred to as excellent in 17 patients (77.3%). FAMM flaps, immediate implants and fixed prostheses enable the functional rehabilitation of oncologic patients, optimizing aesthetics and functional outcomes even in patients undergoing irradiation, thus returning oncologic patients to an excellent quality of life.

Intraosseous Venous Malformation of the Zygomatic Bone: Comparison between Virtual Surgical Planning and Standard Surgery with Review of the Literature.

Intraosseous venous malformations affecting the zygomatic bone are infrequent. Primary reconstruction is usually accomplished with calvarial grafts, although the use of virtual surgical planning, cutting guides and patient-specific implants (PSI) have had a major development in recent years. A retrospective study was designed and implemented in patients diagnosed with intraosseous venous malformation during 2006-2021, and a review of the scientific literature was also performed to clarify diagnostic terms. Eight patients were treated, differentiating two groups according to the technique: four patients were treated through standard surgery with resection and primary reconstruction of the defect with calvarial graft, and four patients underwent resection and primary reconstruction through virtual surgical planning (VSP), cutting guides, STL models developed with CAD-CAM technology and PSI (titanium or Polyether-ether-ketone). In the group treated with standard surgery, 75% of the patients developed sequelae or morbidity associated with this technique. The operation time ranged from 175 min to 210 min (average 188.7 min), the length of hospital ranged from 4 days to 6 days (average 4.75 days) and the postoperative CT scan showed a defect surface coverage of 79.75%. The aesthetic results were "excellent" in 25% of the patients, "good" in 50% and "poor" in 25%. In the VSP group, 25% presented sequelae associated with surgical treatment. The operation time ranged from 99 min to 143 min (average 121 min), the length of hospital stay ranged from 1 to 2 days (average of 1.75 days) and 75% of the patients reported "excellent" results. Postoperative CT scan showed 100% coverage of the defect surface in the VSP group. The multi-stage implementation of virtual surgical planning with cutting guides, STL models and patient-specific implants increases the reconstructive accuracy in the treatment of patients diagnosed with intraosseous venous malformation of the zygomatic bone, reducing sequelae, operation time and average hospital stay, providing a better cover of the defect, and improving the precision of the reconstruction and the aesthetic results compared to standard technique.

Mandibular Reconstruction With Fibula Flap and Dental Implants Through Virtual Surgical Planning and Three Different Techniques: Double-Barrel Flap, Implant Dynamic Navigation and CAD/CAM Mesh With Iliac Crest Graft.

INTRODUCTION: Vertical discrepancy between the fibula flap and the native mandible results in difficult prosthetic rehabilitation. The aim of this study was to evaluate the outcomes of 3D reconstruction of the mandible in oncologic patients using three different techniques through virtual surgical planning (VSP), cutting guides, customized titanium mesh and plates with CAD/CAM technology, STL models and intraoperative dynamic navigation for implant placement. Material and methods. MATERIAL AND METHODS: Three different techniques for mandibular reconstruction and implant rehabilitation were performed in 14 oncologic patients. Five patients (36%) underwent VSP, cutting guides, STL models and a customized double-barrel titanium plate with a double-barrel flap and immediate implants. In six patients (43%), VSP, STL models and a custom-made titanium mesh (CAD/CAM) for 3D reconstruction with iliac crest graft over a fibula flap with deferred dental implants were performed. Three patients (21%) underwent VSP with cutting guides and customized titanium plates for mandibular reconstruction and implant rehabilitation using intraoperative dynamic navigation was accomplished. Vertical bone reconstruction, peri-implant bone resorption, implant success rate, effects of radiotherapy in vertical reconstruction, bone resorption and implant failure, mastication, aesthetic result and dysphagia were evaluated. RESULTS: Significant differences in bone growth between the double-barrel technique and iliac crest graft with titanium mesh technique were found (p<0.002). Regarding bone resorption, there were no significant differences between the techniques (p=0.11). 60 implants were placed with an osseointegration rate of 91.49%. Five implants were lost during the osseointegration period (8%). Peri-implant bone resorption was measured with a mean of 1.27 mm. There was no significant difference between the vertical gain technique used and implant survival (p>0.385). Implant survival rates were higher in non-irradiated patients (p<0.017). All patients were rehabilitated with a fixed implant-supported prosthesis reporting a regular diet (80%), normal swallowing (85.7%) and excellent aesthetic results. CONCLUSIONS: Multi-stage implementation of VSP, STL models and cutting guides, CAD/CAM technology, customized plates and in-house dynamic implant navigation for mandibular defects increases bone-to-bone contact, resolves vertical discrepancy and improves operative efficiency with reduced complication rates and minimal bone resorption. It provides accurate reconstruction that optimizes implant placement, thereby improving facial symmetry, aesthetics and function.

Cervical Lymphadenopathies as Unusual Presentations of Erdheim-Chester Disease: The Need for Knowledge for Diagnosis and Treatment.

The appearance of cervical adenopathies can occur in many pathologies in a non-specific manner; Erdheim-Chester disease (ECD) is characterized by xanthogranulomatous and xanthomatous infiltration of different tissues with numerous foamy histiocytes. Bone lesions are frequent and radiological features are pathognomonic for diagnosis, but lymph node involvement is exceptional and is not a form of presentation reported in the literature. Recurrent BRAFV600E mutation and others have been discovered in recent years. Since then, several treatments targeting the BRAF and MEK pathways have been developed with high success rates; even so, interferon-α continues to be one of the most widely used treatments. The best imaging test for the study and monitoring of the disease is PET-CT. The prognosis of ECD is relatively poor, with a survival of 43% of patients after 32 months follow-up. Higher survival rates have been reported in patients treated with interferon. The authors present an exceptional case of ECD with cervical adenopathies as a debut, highlighting the need for the knowledge of the disease for differential diagnosis, early treatment, and the importance of communication between the clinician and the pathologist. The main features of the disease and a brief discussion of current diagnosis and treatment are reviewed.

Virtual Surgical Planning, Stereolitographic Models and CAD/CAM Titanium Mesh for Three-Dimensional Reconstruction of Fibula Flap with Iliac Crest Graft and Dental Implants.

Mandibular reconstruction with fibula flap shows a 3D discrepancy between the fibula and the remnant mandible. Eight patients underwent three-dimensional reconstruction of the fibula flap with iliac crest graft and dental implants through virtual surgical planning (VSP), stereolitographic models (STL) and CAD/CAM titanium mesh. Vertical ridge augmentation and horizontal dimensions of the fibula, peri-implant bone resorption of the iliac crest graft, implant success rate and functional and aesthetic results were evaluated. Vertical reconstruction ranged from 13.4 mm to 10.1 mm, with an average of 12.22 mm. Iliac crest graft and titanium mesh were able to preserve the width of the fibula, which ranged from 8.9 mm to 11.7 mm, with an average of 10.1 mm. A total of 38 implants were placed in the new mandible, with an average of 4.75 ± 0.4 implants per patient and an osseointegration success rate of 94.7%. Two implants were lost during the osseointegration period (5.3%). Bone resorption was measured as peri-implant bone resorption at the mesial and distal level of each implant, with a variation between 0.5 mm and 2.4 mm, and with a mean of 1.43 mm. All patients were rehabilitated with a fixed implant prosthesis with good aesthetic and functional results.

Vertical Ridge Augmentation of Fibula Flap in Mandibular Reconstruction: A Comparison between Vertical Distraction, Double-Barrel Flap and Iliac Crest Graft.

Double-barrel flap, vertical distraction and iliac crest graft are used to reconstruct the vertical height of the fibula. Twenty-four patients with fibula flap were reconstructed comparing these techniques (eight patients in each group) in terms of height of bone, bone resorption, implant success rate and the effects of radiotherapy. The increase in vertical bone with vertical distraction, double-barrel flap and iliac crest was 12.5 ± 0.78 mm, 18.5 ± 0.5 mm, and 17.75 ± 0.6 mm, (p < 0.001). The perimplant bone resorption was 2.31 ± 0.12 mm, 1.23 ± 0.09 mm and 1.43 ± 0.042 mm (p < 0.001), respectively. There were significant differences in vertical bone reconstruction and bone resorption between double-barrel flap and vertical distraction and between iliac crest and vertical distraction (p < 0.001). The study did not show significant differences in implant failure (p = 0.346). Radiotherapy did not affect vertical bone reconstruction (p = 0.125) or bone resorption (p = 0.237) but it showed higher implant failure in radiated patients (p = 0.015). The double-barrel flap and iliac crest graft showed better stability in the height of bone and less bone resorption and higher implant success rates compared with vertical distraction. Radiation therapy did not affect the vertical bone reconstruction but resulted in a higher implant failure.

Sources of abnormal EEG activity in spontaneous intracerebral hemorrhage.

UNLABELLED: This report describes the results obtained with EEG source analysis in the frequency domain (FD-VARETA), in 14 patients with brain hemorrhages; 6 hemorrhages were located in the putaminal region, 1 was mesencephalic and 7 were lobar cerebral hemorrhages. Our goal was to evaluate FD-VARETA accuracy for the localization of fast growth expansive brain lesions. FD-VARETA produces brain electromagnetic tomography images of EEG sources in every frequency. The location of the most abnormal or the maximum Z value across all frequencies was compared with the location of spontaneous hemorrhages in computed tomographies (CT). In all patients the main source was within delta or theta bands. The spatial extent of the sources, in the brain atlas, at these frequency values was almost the same as the volume of hematoma in CT. Putaminal lesions produced larger regions of cortical deafferentation than lobar hematomas, with higher abnormal Z values. FD-VARETA was more accurate in locating the lesions than traditional maps of absolute and relative power in delta, theta, alpha and beta bands. CONCLUSION: FD-VARETA is a valuable procedure for the functional evaluation of brain hemorrhages.

Migrânea e migrânea "occipital" - Terapia preventiva / Migraine and migraine "occipital" - Prevent therapy

Introdução: Migrânea é, de acordo com a Organização Mundial de Saúde, a 19ª moléstia que mais incapacita o ser humano, sendo desencadeada por vários fatores, com prevalência de 15% no Brasil, e representa 55% das consultas neurológicas. Métodos: Estudo longitudinal prospectivo de coorte desenvolvido durante 3 anos e 7 meses em pacientes com diagnóstico de migrânea, com ou sem aura, em Posto de Saúde Pública de Araxá - MG, utilizando-se o extrato hidroalcoólico de Lippia alba (quimiotipo geraniol-carvenona) preventivo de migrânea. Resultados: Das 8.140 primeiras consultas realizadas nesse período, 545 dos indivíduos (6,7%) apresentaram migrânea, sendo 84% em mulheres. A média de idade foi de 37,02 anos. A sintomatologia neurológica foi destaque em 13,9% dos pacientes e 3,7% dos avaliados pensaram em suicídio. A resposta positiva ao tratamento foi observada em 62,5% dos pacientes e em 37,5% o resultado foi parcial, com redução da intensidade, duração e frequência da dor em mais de 60% dos casos. Conclusão: Além de seu baixo custo e nenhum efeito adverso, o uso do extrato hidroalcoólico de Lippia alba mostrou-se útil no tratamento preventivo de migrânea. Propõe-se a designação "migrânea occipital" devido aos sintomas particulares e principalmente a participação da artéria occipital durante as crises, afastando a menção da ação dos nervos occipitais. (AU)

Introduction: Migraine is the 19th disease that more incapacity the human being according World Health Organization (WHO), trigged by several factors with an amount of 15% in Brazil which represents 55% of neurological appointments. Methods: A prospective longitudinal study of the cohort developed in 3 years and 7 months in patients diagnosed with migraine, with or without aura, in public health agencies in Araxá, using the Lippia alba hydroalcoholic as a preventive from migraine. Results: Among the first 8.140 appointments in this period, 545 of them (6.7%) presented migraine, in which 84% was female and 16% male. The average age was of 37.02. The research showed that 13.9% had featured neurological symptoms and 3.7% from them had suicidal thoughts. 62.5% had positive response with the treatment and in 37.5% there was a partial result with the reduction of intensity, duration and pain relief in more than 60% of the patients. Conclusion: The use of Lippia alba hydroalcoholic extract is useful in migraine preventing treatment besides its low cost and no adverse effect. It is also proposed here the term "migranea occipital" due to the particular symptoms and mainly the occipital artery participation during the crisis, away from the occipital nerves. (AU)

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine.

BACKGROUND: There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients. METHODS: Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry. RESULTS: We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported. CONCLUSIONS: Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.

Rsc14-controlled expression of MNN6, MNN4 and MNN1 regulates mannosylphosphorylation of Saccharomyces cerevisiae cell wall mannoproteins.

ybl006cDelta was selected using an Alcian-blue-based screen aimed to identify nonessential genes involved in the regulation of mannosylphosphorylation. When cells of this deletant were mixed with the cationic dye Alcian blue in a typical assay, they remained white, indicating a low number of mannosylphosphate groups on the cell surface. ybl006cDelta cells did not show any defect in growth rate nor in the glycosylation or secretion rate of the major exoglucanase Exg1. Transcriptome analysis of ybl006cDelta using macroarrays showed at least two-fold changes in the expression of 52 genes (<0.9% of the genome). Three of these have previously been reported to be directly (MNN6 and MNN4) or indirectly (MNN1) implicated in the transfer of mannosylphosphate to the N- and O-oligosaccharides. Alterations in the expression of these genes were confirmed by Northern analysis. YBL006C product was recently identified as a subunit (Rsc14) of the RSC chromatin-remodelling complex of Saccharomyces cerevisiae. It follows that remodelling of chromatin can be an important regulatory mechanism for the maturation of cell wall mannoproteins.

Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoproteins.

We have carried out a screen of 622 deletion strains generated during the EUROFAN B0 project to identify non-essential genes related to the mannosylphosphate content of the cell wall. By examining the affinity of the deletants for the cationic dye alcian blue and the ion exchanger QAE-Sephadex, we have selected 50 strains. On the basis on their reactivity (blue colour intensity) in the alcian blue assay, mutants with a lower phosphate content than wild-type cells were then arranged in groups defined by previously characterized mutants, as follows: group I (mnn6), group II (between mnn6 and mnn9) and group III (mnn9). Similarly, strains that behaved like mnn1 (i.e. a blue colour deeper than wild-type) were included in group VI. To confirm the association between the phenotype and a specific mutation, strains were complemented with clones or subjected to tetrad analysis. Selected strains were further tested for extracellular invertase and exoglucanase. Within groups I, II and III, we found some genes known to be involved in oligosaccharide biosynthesis (ALG9, ALG12, HOC1), secretion (BRE5, COD4/COG5, VPS53), transcription (YOL072w/THP1, ELP2, STB1, SNF11), cell polarity (SEP7, RDG1), mitochondrial function (YFH1), cell metabolism, as well as orphan genes. Within group VI, we found genes involved in environmentally regulated transduction pathways (PAL2 and RIM20) as well as others with miscellaneous or unknown functions. We conclude that mannosylphosphorylation is severely impaired in some deletants deficient in specific glycosylation/secretion processes, but many other different pathways may also modulate the amount of mannosylphosphate in the cell wall.

A search for hyperglycosylation signals in yeast glycoproteins.

N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of alpha-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.