Seismic surveys reduce cetacean sightings across a large marine ecosystem.

Noise pollution is increasing globally, and as oceans are excellent conductors of sound, this is a major concern for marine species reliant on sound for key life functions. Loud, impulsive sounds from seismic surveys have been associated with impacts on many marine taxa including mammals, crustaceans, cephalopods, and fish. However, impacts across large spatial scales or multiple species are rarely considered. We modelled over 8,000 hours of cetacean survey data across a large marine ecosystem covering > 880,000 km2 to investigate the effect of seismic surveys on baleen and toothed whales. We found a significant effect of seismic activity across multiple species and habitats, with an 88% (82-92%) decrease in sightings of baleen whales, and a 53% (41-63%) decrease in sightings of toothed whales during active seismic surveys when compared to control surveys. Significantly fewer sightings of toothed whales also occurred during active versus inactive airgun periods of seismic surveys, although some species-specific response to noise was observed. This study provides strong evidence of multi-species impacts from seismic survey noise on cetaceans. Given the global proliferation of seismic surveys and large propagation distances of airgun noise, our results highlight the large-scale impacts that marine species are currently facing.

Prognostic implications of securin expression and sub-cellular localization in human breast cancer.

PURPOSE: Securin belongs to a class of cell cycle regulators that prevent metaphase-to-anaphase transition until sister chromatid separation is complete. Evidence is accumulating that securin has a prognostic impact on a variety of malignancies but, thus far, the role and regulation of securin expression and its sub-cellular localization have not been systematically addressed in breast cancer. METHODS: In total 470 breast cancer specimens with follow-up data for up to 22 years were included. Immunohistochemical staining and immunofluorescence double-staining were performed for securin and its regulating proteins PTTG1IP, CDC20 and BUBR1. Prognostic associations were evaluated between the expression patterns of these proteins and established prognosticators of invasive breast cancer and patient survival. RESULTS: We found that a high fraction of securin expressing cancer cells predicted an unfavorable clinical outcome of the breast cancer patients (p < 0.001). Also in multivariate analyses, the fraction of securin expressing cancer cells served as an independent prognosticator of a poor survival (p < 0.0001). We also found that the sub-cellular localization of securin exhibited prognostic power, since cytoplasmic securin expression in the cancer cells appeared to be associated with aggressive breast cancer subtypes and high breast cancer-associated mortality rates (p = 0.003). Through immunofluorescence double-staining, we found that PTTG1IP, CDC20 and BUBR1 exhibited distinct patterns of co-expression with securin, suggesting a regulatory role in the metaphase-to-anaphase transition in human breast cancer cells. We also noted that a subgroup of triple-negative breast carcinomas exhibited deviant expression patterns for the proteins studied. CONCLUSIONS: Our data indicate that securin expression may serve as a strong and independent prognosticator of breast cancer outcome and that a cytoplasmic localization of the protein may provide additional prognostic information, particularly in the biologically and clinically challenging subgroup of triple-negative breast carcinomas. The sub-cellular localization of securin appears to reflect the expression of PTTG1IP, CDC20 and BUBR1, which may participate in the regulation of securin activity and, ultimately, in the survival of breast cancer patients.

Altered expression of p120catenin predicts poor outcome in invasive breast cancer.

PURPOSE: The study focuses on p120catenin, a regulator of cell adhesion, which has previously been described in many malignancies and suggested with a role in invasion and metastatic behaviour. In this study, we investigate the role of altered immunoexpression of p120catenin isoforms in the prognosis of invasive breast cancer (n = 351). METHODS: We used cDNA microarrays to screen differences in gene expression in invasive breast cancer in general, and between local and metastasized disease particularly. On this basis, we performed p120catenin immunohistochemistry in order to confirm the prognostic value of p120catenin isoforms on tissue microarrays comprising 341 patients from the era of mammographic screening, directed to modern surgical and oncological treatments, and followed-up for maximum of 20 years. RESULTS: In cDNA microarray analysis, p120catenin was discovered down-regulated along with E-cadherin and alpha-catenin. In addition, p120catenin distinguished metastasized breast cancer from local disease. Immunohistochemistry confirmed the value of p120catenin as an independent prognosticator of breast cancer survival. In our results, p120catenin was associated with 3.7-fold risk of breast cancer death in multivariate Cox's regression analyses adjusted for the established prognosticators of breast cancer (p = 0.039). Particularly, the long isoform of p120catenin predicted metastatic disease (p = 0.029). CONCLUSION: The present paper is the first report on p120catenin in invasive breast cancer based on a well-characterized patient material with long-term follow-up. We observed altered expression of p120catenin isoforms in invasive breast cancer and, in our material, the decrease in p120 immunoexpression was significantly associated with poor outcome of disease.

Securin promotes the identification of favourable outcome in invasive breast cancer.

BACKGROUND: Securin is a recently recognised oncogene with multiple known functions in initiation, progression and cell cycle regulation in several malignant diseases, including breast carcinoma. METHODS: In this paper, the prognostic value of securin is evaluated by immunohistochemistry in 310 patients diagnosed with invasive breast cancer during a mammographic screening programme in Central Finland. All patients were directed to modern surgical and oncological treatments and were followed up for a maximum of 20 years. RESULTS: Our results suggest that securin immunopositivity is an independent prognosticator of invasive breast cancer. In our study, securin predicted breast cancer-specific survival among all cases of invasive breast cancer and subgroups divided according to histological type, Ki-67 proliferation status and tumour size. Especially in a multivariate analysis standardised for axillary lymph node status, patient's age and tumour size at the time of diagnosis, securin immunopositivity indicated a 13.1-fold risk of breast cancer death (P=0.024) among invasive ductal breast carcinomas with low Ki-67 positivity. CONCLUSION: Our present and previous results suggest that securin could be useful in clinical pathology to intensify the power of the established prognosticators of invasive breast cancer and, especially, to assist in identifying patients with a more favourable outcome than that indicated by Ki-67 alone.

Neck muscle training in the treatment of chronic neck pain: a three-year follow-up study.

AIM: The objective of the trial was to evaluate whether the positive results achieved with a one-year training regimen in patients with chronic nonspecific neck pain would have long-standing effects. METHODS: A follow-up study of two neck muscle training groups after a randomized controlled study was carried out. One-hundred and eighteen women included were those who had performed neck strength and endurance exercises in a previous randomised controlled trial. The primary outcome measures were neck pain measured by the visual analogue scale and disability indices. Isometric neck strength, range of motion (ROM) and pressure pain threshold (PPT) were measured and training frequency for the previous month elicited by a questionnaire. RESULTS: At the 3-year follow-up, neck pain and the disability indices showed no statistically discernible change compared to the situation at the 12-month follow-up. Also, gains in neck strength, ROM and PPT achieved during the training year were largely maintained. However, adherence to the specific home training program faltered considerably. CONCLUSION: The improvements achieved through long-term training were maintained at the 3-year follow-up. Since a 12-month exercise programme shows a long-term effect, exercise may not need to be performed regularly for the remainder of the subject's life.

Localization of M2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods.

We investigated parvalbumin (PV) and calretinin (CR) containing interneurons in the rat entorhinal cortex. RNA amplification following single cell dissection of immunohistochemically labeled cells from layers II to VI revealed that PV cells, in contrast to CR cells, express the m2 muscarinic receptor (M2AchR) protein. Double immunostaining to confirm the results of RNA amplification indicated that the majority of PV cells contain M2AchR protein, whereas only a small proportion of CR cells do. In contrast, a large number of layer I CR cells, which are mostly Cajal-Retzius cells, were positive for M2AchR. RNA amplification following dissection of these cells also revealed that they contain the M2AchR protein. These findings emphasize that there are significant differences in the expression of different proteins, even among similar neuronal types in the same brain region. This highlights the importance of accurately collecting single cells, and knowledge of anatomical details in molecular biological studies.

Predicting aggressive outcome in T1N0M0 breast cancer.

Despite the excellent overall prognosis, unpredictable breast cancer recurrences and deaths also occur among T1N0M0 patients. We have evaluated clinically applicable methods for identifying aggressive outcome in T1N0M0 breast cancer. The material is based on aggressive T1N0M0 invasive ductal and lobular carcinomas diagnosed in Turku University Hospital and Jyväskylä Central Hospital, Finland, during 1987-1997. We studied all the T1N0M0 breast cancers that had led to recurrency or death (n=21, 95% T1cN0M0) during the follow-up period (4-14 years). The study is based on statistical analyses of matched case-control data in which the prognostic factors of each individual patient with aggressive disease were compared with control patients (n=45) individually matched by tumour size, age at diagnosis, histological type of tumour and length of follow-up. The cancer cases were examined for clinically applicable conventional and immunohistochemical pathologic prognostic factors. High Ki-67 immunopositivity was the strongest prognosticator of breast cancer death or recurrence in T1N0M0 breast cancer. Also, high p53 immunopositivity, low oestrogen receptor immunopositivity and Her-2/neu oncogene amplification by chromogen in situ hybridisation were reliable indicators of unfavourable outcome. Our statistical methods also allowed us to determine for the present material a range of clinical significance for each immunohistochemical prognostic feature with the associated relative risk for breast cancer death and recurrence. The paper suggests guidelines for predicting aggressive outcome in T1N0M0 breast cancer.

Purification of filaggrin from human epidermis and measurement of antifilaggrin autoantibodies in sera from patients with rheumatoid arthritis by an enzyme-linked immunosorbent assay.

BACKGROUND: The so-called antikeratin antibody (AKA) and the antiperinuclear factor (APF) that recognize proteins related to human epidermal filaggrin belong to the most specific serological markers of rheumatoid arthritis (RA). However, assays for the detection of AKA and APF are currently based on immunofluorescence, a method that is subject to arbitrary interpretation and inadequate standardization of the substrates. METHODS: Proteins extracted from human epidermis were separated by reversed-phase high-performance liquid chromatography (HPLC). Filaggrin-containing fractions, identified in immunoblotting by monoclonal antifilaggrin antibodies, were then subjected to gel filtration HPLC and, finally, to a second reversed-phase HPLC step. Tryptic digestion, amino acid sequencing and mass spectrometry were used to confirm the identity of the purified protein. Filaggrin was used as antigen in enzyme-linked immunosorbent assay (ELISA) to measure IgG class antifilaggrin antibodies. RESULTS: The filaggrin preparation obtained gave a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, binding monoclonal antifilaggrin antibody in immunoblotting. Amino acid sequences of all 10 tryptic peptides analyzed were shown to originate from human filaggrin. Antifilaggrin antibody levels exceeded the 99th percentile level of 100 middle-aged blood donors in 26/55 (47%) RA sera. At a similar cutoff level 28/55 (51%) of the RA sera were positive in the AKA test. Of the 26 antifilaggrin-positive sera, 21 were also AKA-positive. CONCLUSION: Human filaggrin can be purified by standard biochemical techniques, despite the heterogeneity of the protein, and used in ELISA for testing autoantibodies to filaggrin. The sensitivity of the assay equals that of the AKA test.

Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei.

Localization of expression and secretion of a heterologous barley cysteine endopeptidase (EPB) and the homologous main cellobiohydrolase I (CBHI) in a Trichoderma reesei transformant expressing both proteins were studied. The transformant was grown on solid medium with Avicel cellulose and lactose to induce the cbh1 promoter for the synthesis of the native CBHI and the recombinant barley protein linked to a cbh1 expression cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirect immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher molecular weight than the native barley protein, probably due to glycosylation and differential processing in the fungal host. As was found with its transcripts, recombinant EPB was localized in apical and subapical compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI was also secreted from these. In immunoelectron microscopy, the endoplasmic reticulum and spherical vesicles assumed to contribute to secretion were labeled by both CBHI and EPB antibodies while only CBHI was localized in elongated vesicles close to the plasma membrane and in hyphal walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins.

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.

Verbal autopsy as a technique to establish causes of infant and child mortality.

In order to develop a method for retrospectively establishing causes of paediatric deaths in Mangochi District, Malawi, we translated a verbal autopsy questionnaire into Chichewa and Chiyao. This form was used to interview guardians of 82 children who died in hospital between June 1993 and May 1994. Based on the guardians' answers, we were able to determine the causes of death for these children. According to our experience, verbal autopsy appeared simple to administer and relatively easy to interpret. To test the validity of this method in southern Malawi, we compared its results to hospital diagnosis, using a sample of 36 children who had died in hospital and whose records could be located. In this preliminary validation, we found a high sensitivity and specificity for the most common childhood illnesses in the area. Thus, verbal autopsy appeared a promising method of collecting population-based information about paediatric mortality, the full applicability of which should be tested in larger studies in Malawi.

Pulsed ultrasound treatment of the painful shoulder a randomized, double-blind, placebo-controlled study.

To study the effect of pulsed ultrasound in shoulder pains, 35 patients were treated with pulsed ultrasound and 37 patients with placebo ultrasound in a double-blind design. The therapy was given during inpatient rehabilitation, 10-12 treatments over 3-4 weeks. Treatment time was 10 minutes, frequency 1.0mHz, on-off ratio 1:4 and intensity 1.0w/cm2. Follow-ups were done after 4-12 months. No differences (p < 0.05) in outcomes were found between the groups after the treatment period or at follow-ups. These results discourage the adding of pulsed ultrasound therapy with the variables used to the conservative treatment of the painful shoulder.

Improved immunoturbidimetric method for rheumatoid factor testing.

The performance of two immunoturbidimetric modifications for rheumatoid factor (RF) testing, which differ with respect to the means of complement inactivation (heat treatment and inactivation with polyvinyl sulphonate), were compared in serum samples from 87 patients with rheumatoid arthritis (RA) and from 403 healthy subjects. IgM-rheumatoid factor titres were also measured with an enzyme linked immunosorbent assay (ELISA). Both immunoturbidimetric tests gave positive reactions (rheumatoid factor > or = 20 IU/ml) in 74 out of the 87 (85%) RA sera. In cases with high RF concentrations the results after chemical inactivation tended to be slightly higher compared with heat inactivation. In healthy subjects rheumatoid factor was detected in 19/403 (4.7%) sera using heat inactivation and in 22/403 (5.5%) sera with chemical inactivation of complement. Interrun coefficient of variation in the chemical inactivation assay was 4.4%; with the heat inactivation method it was 8.1%. In the ELISA, a marginally better correlation was noted in the results obtained using chemical inactivation. Inactivation of complement by means of polyvinyl sulphonate offers the advantage of easier test performance and better reproducibility, and the results may reflect more accurately true rheumatoid factor concentrations.

On the microscopic anatomy of the rete testis a scanning electron microscopic and light microscopic study.

The microscopic anatomy of the transitional zone of the seminiferous tubules, the tubuli recti and the rete testis in adult rats was studied with histological serial sections, semithin sections and scanning electron microscopy. In paraffin section most transitional zones of the seminiferous tubules seemed to be obliterated by the modified Sertoli cells. Thinner plastic sections showed always a lumen, however. PAS--positive material, thought to represent masses of degenerating spermatozoa surrounded by Sertoli cell nuclei was found in 20% of transitional zones. About 80% of the tubuli recti had an initial widening which surrounded the bulging Sertoli cell bodies of the transitional zones. The intratunical rete consisted of five to seven intercommunicating channels, usually of small caliber. One wide communication was regularly present, however. The extratesticular rete was usually formed of two wide cavities. From their subdivisions the five to seven ductuli efferentes arose. The rete epithelium varied from very thin squamous to cuboidal and even columnar. The epithelial cells contained a flagellum surrounded by peripheral microvilli. Loose connective tissue was found under the epithelium of all parts of the rete.

Morphology of the rat rete testis in experimental autoimmune orchitis.

The role of the rete testis and related structures in the development of experimental autoimmune orchitis (EAO) was studied in adult inbred Sprague-Dawley rats. Histology and electron microscopy showed focal perivascular infiltration and an increase in the number of mast cells and polymorphonuclears. Often there were vacuolated macrophages. These changes were first observed two weeks after the beginning of immunization, but they did not clearly precede lesions in seminiferous tubules. Increased amounts of cellular debris appeared in rete cavities, suggesting damage to germinal epithelium. Lanthanum tracer studies of immunized rats showed local lesions in the blood-testis barrier of the seminiferous tubules but did not indicate spreading of lesions from the rete testis. Efferent duct ligation of immunized rats for 24 h did not increase the frequency of lesions. The present observation do not support the theory that the rete testis is the route of spreading of EAO.

Loose connective tissue of rat rete testis. Fine structure, postnatal development and effect of efferent duct ligation.

Fine structure, postnatal development and reaction to efferent duct ligation of the loose connective tissue of the rat rete testis were studied by light and electron microscopy. The loose connective tissue of adult rats consists of elongate fibroblasts in a homogenous ground substance, together with some Leydig cells, lymphocytes, macrophages and mast cells. During postnatal development this tissue increases in amount, while the interstitial areolar tissue decreases. The "looseness" of the tissue becomes more evident between days 22 and 27, and may reflect an increase in hydration. Efferent duct ligation for 15 min to five days has no effect on the histological appearance of the tissue.

Fine structure of the transitional zone of the rat seminiferous tubule.

An electron microscopic study was made on the structure of the testicular transitional zone (TZ) in the adult rat. The TZ proper consists of modified Sertoli cellss, with only a few spermatogonia and macrophages, surrounding distally a very narrow lumen. The TZ Sertoli cells have nuclei with a somewhat coarser matrix and more peripheral heterochromatin than Sertoli cell nuclei of the nearby seminiferous tubules, and the electron density of the cytoplasm varies from cell to cell. Smooth endoplasmic reticulum is abundant, but usually there are also scattered ribosomal rosettes and an occasional profile of rough endoplasmic reticulum. Microtubules are very numerous in the columnar portion of the cell, and laminar structures seemingly joining the cell surfaces are sometimes seen. Lipid droplets and lysosmal structures are frequent cellular components in proximal TZ Sertoli cells. Empty intracellular vacuoles are abundant, sometimes arranged around areas of smooth endoplasmic reticulum. Occasionally, membrane-limited fine granules and vacuoles are seen within Sertoli cells and also in the TZ lumen, suggesting a possible secretory activity by these cells. The apical processes of the Sertoli cells form large vacuolar structures, and in the basal parts of the epithelium vacuoles with capillary-like appearance are frequently seen. Phagocytosis of germinal cells by the Sertoli cells occurs in the proximal region of the TZ. Round waste bodies in contact with the Sertoli cell apices protruding into the tubulus rectus, are also common. The tunica propria of the TZ is thickened and somewhat wrinkled, and in the proximal region the myoid cell layer loses its continuity and is replaced by fibroblasts. The epithelium of the tubulus rectus adjacent to the TZ consists of several overlapping epithelial cells. The typical junctional complexes between TZ Sertoli cells appear to be impermeable to the lanthanum tracer.