Methanomethylovorans are the dominant dimethylsulfide-degrading methanogens in gravel and sandy river sediment microcosms.

BACKGROUND: Rivers and streams are important components of the global carbon cycle and methane budget. However, our understanding of the microbial diversity and the metabolic pathways underpinning methylotrophic methane production in river sediments is limited. Dimethylsulfide is an important methylated compound, found in freshwater sediments. Yet, the magnitude of DMS-dependent methanogenesis nor the methanogens carrying out this process in river sediments have been explored before. This study addressed this knowledge gap in DMS-dependent methanogenesis in gravel and sandy river sediments. RESULTS: Significant methane production via DMS degradation was found in all sediment  microcosms. Sandy, less permeable river sediments had higher methane yields (83 and 92%) than gravel, permeable sediments (40 and 48%). There was no significant difference between the methanogen diversity in DMS-amended gravel and sandy sediment microcosms, which Methanomethylovorans dominated. Metagenomics data analysis also showed the dominance of Methanomethylovorans and Methanosarcina. DMS-specific methyltransferase genes (mts) were found in very low relative abundances whilst the methanol-, trimethylamine- and dimethylamine-specific methyltransferase genes (mtaA, mttB and mtbB) had the highest relative abundances, suggesting their involvement in DMS-dependent methanogenesis. CONCLUSIONS: This is the first study demonstrating a significant potential for DMS-dependent methanogenesis in river sediments with contrasting geologies. Methanomethylovorans were the dominant methylotrophic methanogen in all river sediment microcosms. Methyltransferases specific to methylotrophic substrates other than DMS are likely key enzymes in DMS-dependent methanogenesis, highlighting their versatility and importance in the methane cycle in freshwater sediments, which would warrant further study.

Methanolobus use unspecific methyltransferases to produce methane from dimethylsulphide in Baltic Sea sediments.

BACKGROUND: In anoxic coastal and marine sediments, degradation of methylated compounds is the major route to the production of methane, a powerful greenhouse gas. Dimethylsulphide (DMS) is the most abundant biogenic organic sulphur compound in the environment and an abundant methylated compound leading to methane production in anoxic sediments. However, understanding of the microbial diversity driving DMS-dependent methanogenesis is limited, and the metabolic pathways underlying this process in the environment remain unexplored. To address this, we used anoxic incubations, amplicon sequencing, genome-centric metagenomics and metatranscriptomics of brackish sediments collected along the depth profile of the Baltic Sea with varying sulphate concentrations. RESULTS: We identified Methanolobus as the dominant methylotrophic methanogens in all our DMS-amended sediment incubations (61-99%) regardless of their sulphate concentrations. We also showed that the mtt and mta genes (trimethylamine- and methanol-methyltransferases) from Methanolobus were highly expressed when the sediment samples were incubated with DMS. Furthermore, we did not find mtsA and mtsB (methylsulphide-methyltransferases) in metatranscriptomes, metagenomes or in the Methanolobus MAGs, whilst mtsD and mtsF were found 2-3 orders of magnitude lower in selected samples. CONCLUSIONS: Our study demonstrated that the Methanolobus genus is likely the key player in anaerobic DMS degradation in brackish Baltic Sea sediments. This is also the first study analysing the metabolic pathways of anaerobic DMS degradation in the environment and showing that methylotrophic methane production from DMS may not require a substrate-specific methyltransferase as was previously accepted. This highlights the versatility of the key enzymes in methane production in anoxic sediments, which would have significant implications for the global greenhouse gas budget and the methane cycle. Video Abstract.

The healthcare system and provision of oral healthcare in European Union member states. Part 4: Greece.

This paper presents a description of the healthcare system and how oral healthcare is organised and provided in Greece, a country in a deep economic and social crisis. The national health system is underfunded, with severe gaps in staffing levels and the country has a large private healthcare sector. Oral healthcare has been largely provided in the private sector. Most people are struggling to survive and have no money to spend on general and oral healthcare. Unemployment is rising and access to healthcare services is more difficult than ever. Additionally, there has been an overproduction of dentists and no development of team dentistry. This has led to under or unemployment of dentists in Greece and their migration to other European Union member states, such as the United Kingdom, where over 600 Greek dentists are currently working.

Modeling of single-cell oil production under nitrogen-limited and substrate inhibition conditions.

Sweet sorghum extract was used as substrate for lipid accumulation by the oleaginous fungus Mortierella isabellina in batch cultures. Various initial sugar (13-91 g/L) and nitrogen (100-785 mg/L) concentrations resulting in various C/N (43-53) ratios were tested. Oil accumulation ranged between 43% and 51% corresponding to oil production from 2.2 to 9.3 g/L. A detailed mathematical model was developed. This model is able to adequately predict biomass growth, lipid accumulation, and sugar and nitrogen consumption. The model assumes that fungus growth is inhibited at high sugar concentrations. A set of kinetic experiments was used for model kinetic parameters estimation, while another set of experiments was used for model validation. The developed model could be generalized for similar systems of lipid accumulation and become a useful tool for reactor design for biofuel production.

Fibroadenoma masquerading carcinoma on fine-needle aspiration of the breast.

OBJECTIVE: Benign and malignant lesions of the breast may have similar appearances on fine-needle aspiration cytology. We report a case of fibroadenoma that was diagnosed as carcinoma by cytology. CASE STUDY: Breast fine-needle aspiration biopsy was highly cellular and composed of bland-appearing spindle/columnar cells that could represent either epithelial or stromal cells; the case was reported as positive and the patient had subsequent excisional biopsy taken. RESULTS: On microscopic examination, smears were hypercellular and had many single cells and clusters of columnar/ elongate cells No obvious bipolar cells of myoepithelial origin were seen. Significant atypia was noted. Immunocytochemistry for smooth muscle actin was not performed due to insufficient material. CONCLUSIONS: Some cases of fibroadenoma and carcinoma can be very difficult to distinguish on fine needle aspiration cytology smears. Immunocytochemistry may be of help if sufficient material is provided. To avoid false positive diagnosis on cytology, it is best to report such a case as intermediate (atypical/suspicious) with final interpretation pending excisional biopsy.

Intraoperative touch imprint cytological analysis of sentinel lymph nodes for the presence of metastases in malignant melanoma.

AIM: Sentinel lymph node (SLN) biopsy has revolutionized lymph node staging in patients with malignant melanoma. Intraoperative evaluation is a new addition to the SLN procedure that allows for a one-step regional lymph node dissection to be performed when the SLN biopsy findings are positive. The discriminatory immunostaining pattern with the S-100 and HMB45 monoclonal antibodies allows intraoperative immunocytochemical evaluation of imprint smears of SLNs for melanoma metastases. METHODS: One hundred twenty eight SLNs from a cohort of 52 patient-cases that had been identified using sulfur colloid as a radioactive tracer and isosulfan blue were bisected for rapid Diff-Quick stained touch preparations. Intraopera-tive evaluation of sentinel node status by imprint cytology was correlated with the histopathological results of permanent sections. Tumor-negative nodes in routine paraffin sections were further investigated with the employment of the S-100 and HMB45 antibodies. RESULTS: Thirty-six of all SLNs harbored metastases in paraffin sections, from which 32 were identified by imprint cytology (sensitivity 88.8%). Three SLNs were positive by imprint cytology and negative by histopathology of paraffin sections. Comparison of the results of the touch preparations with the final histopathology (hematoxylin-eosin and S-100/ HMB45 stains) demonstrated a sensitivity of 83.3% and a negative predictive value of 92.5%. The specificity and positive predictive value were 100% respectively. CONCLUSION: Touch imprint cytology is potentially useful for intraoperative evaluation of SLNs in malignant melanoma patients. Results can be improved if the surface sampled is appropriately enlarged and a rapid immunohistochemical S-100/HMB45 stain on the imprints is utilized.

Endometriosis involving the rectus abdominis muscle and subcutaneous tissues: fine needle aspiration appearances.

OBJECTIVE: Endometriosis is defined as functioning endometrial tissue outside the uterine cavity. It occurs in up to 15% of menstruating females and in most cases is located within the pelvis. Endometrial implants, however have been described in soft tissues, particularly in the skin and subjacent tissues of surgical scars, and diagnosis might be problematic. CASE STUDY: A 32 aged female presented with a suprapubic abdominal mass, which appeared suddenly after exercise. Fine needle aspiration was performed. RESULTS: Epithelial sheets were shown in direct aspirates. No evident endometrial stromal cells were seen. CD10 immunostaining in additional cell block preparations using a commercial antibody gave positive results. The cell pattern and immunocytochemical profile suggested a cytodiagnosis of endometriosis. The patient was administered with leuprolide acetate. She experienced adverse effects related to estrogen deficiency. Medical treatment was discontinued and the patient underwent surgical excision. Histological sections revealed endometrial glands surrounded by stroma and embedded in fibrous connective tissue. CONCLUSION: With optimal preparations a confident cytological diagnosis of endometriosis may be established easily, allowing correct treatment of the disease and, in selected cases, planning of preoperative pharmacologic therapy.

Membrane adsorption with direct cell culture combined with reverse transcription-PCR as a fast method for identifying enteroviruses from sewage.

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Alpha9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

Upper airway edema resulting from use of Ecballium elaterium.

OBJECTIVES: To present a rare occurrence in ear, nose and throat practice of upper airway allergic edema from use of juice extracted from the fruit of Ecballium elaterium. INTRODUCTION: Ecballium elaterium is a plant indigenous to the Mediterranean region that bears the common name squirting cucumber. Many people in this region for the treatment of sinusitis use the juice of its fruit. However, allergy-prone patients after using it may sometimes present with edema at various sites of the upper respiratory tract. Otolaryngologists are frequently asked to diagnose and treat such an emergency situation. STUDY DESIGN: Retrospective review of the records of 42 patients with this condition during the last 4 years. METHODS: Patient records were retrospectively reviewed for age, sex, presenting symptoms, physical examination, and medical treatment. Seasonal distribution and data from the history of the patients were also considered. Skin prick and prick-to-prick testing to various allergens was performed in 12 patients. RESULTS: Most of the patients presented with localized swelling of the uvula and the nasal mucosa, whereas in the remainder of the patients various sites of swelling of the upper respiratory tract were observed. Skin tests elicited positive reactions to pollen weeds and to various fruits of the Cucurbitaceae family. Treatment of the patients with corticosteroids and antihistamines resulted in an uneventful recovery, and we did not need to resort to therapeutic modalities, such as intubation or tracheotomy. CONCLUSIONS: We think a further study of the pharmaceutical properties of the plant's fruit juice is needed, especially regarding its curative properties of rhinitis and sinusitis. However, adverse reactions in some patients with a positive history of allergy should always be considered.

Submasseteric abscess.

The masseteric space is an important tissue compartment of the neck, but disease in it is difficult to diagnose and treat. In this paper a case of a young adult male with an abscess of the submasseteric space is presented. Diagnosis was established by computed tomography (CT) of the neck, but the severity of the lesion was not accurately estimated. Surgical intervention was performed and a large quantity of pus was drained. A detailed medical history and clinical examination of the patient as well as CT are important tools in the accurate diagnosis and efficient treatment of the disease.

Periodic alternating nystagmus associated with Arnold-Chiari malformation.

The Arnold-Chiari malformation type I is a rare congenital condition characterized by herniation of the cerebellar tonsils into the upper cervical spinal tract. Various otoneurological manifestations occur in patients with the disease, which has usually been associated with a downbeat type of nystagmus. In this paper a case with this condition is reported presenting with periodic alternating nystagmus (PAN), which is a rare type of nystagmus observed in diseases of the central nervous system of different pathologies. The patient had a complete otoneurological examination and vestibular function testing. Diagnosis was established by using magnetic resonance imaging (MRI).

Effect of the number of averaged responses in transient evoked otoacoustic emissions on the results of neonatal hearing screening.

This study examined the effect of the number of accepted responses in transient evoked otoacoustic emissions on the results of neonatal hearing screening programmes. The ILO88 Otodynamics Analyzer Quickscreen programme was used for all testing, and a three-stage procedure was adopted by averaging 20, 30, and 260 low-noise samples in total. The results were recorded after each stage of the testing in those cases in which, after the first 20 accepted responses, the "pass" criteria were met. Under these circumstances, 117 ears were included in the study from a total number of 334 screened ears. It was concluded that 20 averaged quiet responses are adequate for screened newborn babies to pass the test if the conditions of the "pass" criteria are fulfilled at this stage. In the rest of the newborn babies, testing should be continued using a larger number of clicks. For diagnostic and clinical purposes, the full 260 quiet samples must be used since the results indicated statistically better scores in response and reproducibility measures after the 260 averaged responses.

Expression of MAGE and GAGE in high-grade brain tumors: a potential target for specific immunotherapy and diagnostic markers.

The mRNA expression of the tumor-associated antigens MAGE and GAGE was examined in 60 high-grade brain tumors. This analysis was performed by using reverse transcription-PCR, Southern blotting, and sequencing. It was demonstrated that, of the eight GAGE genes, GAGE-2 and -7 were expressed in five of seven normal brains. Four groups of tumors--adult glioblastoma multiforme (n = 20), pediatric glioblastoma multiforme (n = 9), medulloblastomas (n = 15), and ependymomas (n = 14)--were analyzed for mRNA expression. The following frequencies were observed: MAGE-1, 0, 0, 13, and 0%, respectively; MAGE-2, 5, 11, 60, and 57%; MAGE-3 & -6, 0, 0, 13, and 0%; GAGE-1, 65, 11, 13, and 43%; and GAGE-3-6 and -8: 75, 78, 47, and 93%, respectively. Two unclassified tumors expressed GAGE-3-6 and -8 only. The absence of GAGE-1 expression in normal brain, its relatively high frequency of expression in high-grade brain tumors, and its unique 3' sequence, suggest it may represent a useful target for specific immunotherapy. The detection method of reverse transcription-PCR and Southern blotting may also be useful for rapid screening of biopsy specimens both for diagnostic purposes and to determine a patient's eligibility for specific immunotherapy.

Structure of the gene encoding the murine dual specificity tyrosine-threonine phosphatase PAC1.

The mitogen-induced early-response gene, PAC-1, encodes a nuclear 32-kDa tyrosine-threonine dual specificity phosphatase, which has been shown to specifically dephosphorylate the mitogen activated protein (MAP) kinases, ERK1 and ERK2. Here, we describe the structure and sequence of the murine PAC-1 gene. Transcription starts at three major sites located between 80 and 90 nucleotides upstream of the murine PAC-1 initiation codon within a highly G/C-rich region. The gene comprises three exons, with exon 1 encoding the unique N-terminal half of the protein, while exons 2 and 3 encode the C-terminus that is homologous to the closely related phosphatases, 3CH134 and VH1. The conserved catalytic domain common to all tyrosine phosphatases is encompassed by exon 3. The organization of the murine PAC-1 gene suggests that the PAC-1 N-terminus, which may serve a regulatory function, has evolved as a separate domain from the C-terminal catalytic domain.

Cervicofacial actinomycosis presenting as acute upper respiratory tract obstruction.

An unusual case of cervicofacial actinomycosis presenting as acute upper airway obstruction and demanding urgent tracheostomy is reported. Diagnosis was established by microscopic examination of the pus and culture of Actinomyces israelii. Repeated surgical drainage of the purulent foci and prolonged treatment with penicillin obtained resolution of the disease. Clinicians dealing with acute head and neck swellings should always consider actinomycosis as a possible diagnosis.

Adult-type rhabdomyoma of the submandibular region.

Adult-type rhabdomyoma is a rare benign tumor which occurs mainly in the head and neck region of middle-aged patients. We present a case of rhabdomyoma of the submandibular region. Clinical and histologic features are discussed as well as differential diagnosis and proper treatment. Finally, all previous reports of adult rhabdomyoma cases are reviewed.

Multiple glomus tumours.

A patient with multiple, synchronous, non-familial head and neck paragangliomas is reported. There were three primary neoplasms, a glomus tympanicum and glomus vagale on the right side and a glomus tumour of the carotid body on the left. Such a combination has never been reported previously. The reports of all the series with paragangliomas in the literature, as well as the reports of single cases with multiple tumours during the last three decades, are reviewed. Specific problems in diagnosis and management of multiple glomus tumours are discussed.

Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E.

Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity.

A new quantitative polymerase chain reaction-high performance ion exchange liquid chromatographic method for the detection of fibroblast growth factor-beta (FGF-beta) gene amplification.

A method for the quantitative determination of fibroblast growth factor-beta (FGF-beta) genomic amplification based on the use of optimized polymerase chain reaction (PCR) procedures and high performance ion exchange (HPIEX) liquid chromatography has been developed. Co-amplification of a second genomic species permits internal standardization of the techniques during optimization of the reaction conditions, the PCR cycle number and the PCR cycle efficiency, as well as during the analytical HPIEX chromatographic determination of the PCR products. These investigations confirm the versatility of these procedures to quantitatively analyse FGF-beta gene amplification in various cells and tissues.

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.