Characterisation of spatial variability in water quality in the Great Barrier Reef catchments using multivariate statistical analysis.

Water quality monitoring is important to assess changes in inland and coastal water quality. The focus of this study was to improve understanding of the spatial component of spatial-temporal water quality dynamics, particularly the spatial variability in water quality and the association between this spatial variability and catchment characteristics. A dataset of nine water quality constituents collected from 32 monitoring sites over a 11-year period (2006-2016), across the Great Barrier Reef catchments (Queensland, Australia), were evaluated by multivariate techniques. Two clusters were identified, which were strongly associated with catchment characteristics. A two-step Principal Component Analysis/Factor Analysis revealed four groupings of constituents with similar spatial pattern and allowed the key catchment characteristics affecting water quality to be determined. These findings provide a more nuanced view of spatial variations in water quality compared with previous understanding and an improved basis for water quality management to protect nearshore marine ecosystem.

Hyperostosis frontalis interna in female historic skeletal populations: Age, sex hormones and the impact of industrialization.

OBJECTIVES: This analysis aims to investigate the impact of industrialization on the prevalence of Hyperostosis Frontalis Interna (HFI), focusing on the roles of age and parity to examine the claim that longevity and changing reproductive patterns have led to increased rates in modern populations. MATERIALS AND METHODS: A total of 138 individuals from two documented London skeletal assemblages of the Industrial period were analyzed employing macroscopic observation, digital radiography and MicroCT scanning to establish the prevalence rates of HFI according to modern clinical standards. Statistical analysis was also undertaken on a sub-sample of 51 females of post-menopausal age to identify any relationship between parity and HFI. RESULTS: The majority of cases of HFI were found in older females, reflecting clinical observations. The prevalence rates of HFI corresponded well to those predicted from the proportion of old age females present within populations. Age was therefore shown to be a predominant factor in HFI presence. A plateau in HFI prevalence was noted from the age of 50-59 years onwards. No statistically significant relationship was found between parity and HFI. DISCUSSION: When recorded consistently, HFI was positively correlated with age and longevity but had also increased among old age females over time. Our results suggest that nulliparity co-occurs with HFI but is not a primary factor in its pathogenesis. Key factors in HFI presence in females are likely to be increased androgens and the dysregulation of insulin and insulin-like growth factor-1.

Digital radiography and historical contextualisation of the 19th century modified human skeletal remains from the Worcester Royal Infirmary, England.

Recent excavations at the Worcester Royal Infirmary revealed a large assemblage of modified skeletal remains dating to the late 19th century. The assemblage included a sample of 134 long bones, 76 of which were transected and 58 of which displayed evidence of gross pathology without transection. Transection may have been undertaken for the purposes of amputation, anatomical or morbid dissection, for surgical training or specimen preparation. In order to elucidate the motives for such modification, Direct Digital Radiography (DDR) was undertaken on the proximal and distal elements present to confirm the true prevalence of disease. The radiographic evidence revealed that some transected elements exhibited previously unobserved pathological changes. The ratios of proximal to distal elements varied between samples of pathological and non-pathological transected elements, suggesting different motives for modification. There was also a significant difference between the composition of the skeletal sample and that expected from historical records of amputation. Aggressive or acute/subacute as well as chronic stages of disease were observed, some lesions possibly relating to ascending infection following gangrenous limb ischaemia or haematogenous spread. Other rare conditions were detected using DDR, proving it to be a critical tool in the assessment of skeletal disease in the past.

Morbidity in the marshes: using spatial epidemiology to investigate skeletal evidence for Malaria in Anglo-Saxon England (AD 410-1050).

Concerns over climate change and its potential impact on infectious disease prevalence have contributed to a resurging interest in malaria in the past. A wealth of historical evidence indicates that malaria, specifically Plasmodium vivax, was endemic in the wetlands of England from the 16th century onwards. While it is thought that malaria was introduced to Britain during the Roman occupation (AD first to fifth centuries), the lack of written mortality records prior to the post-medieval period makes it difficult to evaluate either the presence or impact of the disease. The analysis of human skeletal remains from archaeological contexts is the only potential means of examining P. vivax in the past. Malaria does not result in unequivocal pathological lesions in the human skeleton; however, it results in hemolytic anemia, which can contribute to the skeletal condition cribra orbitalia. Using geographical information systems (GIS), we conducted a spatial analysis of the prevalence of cribra orbitalia from 46 sites (5,802 individuals) in relation to geographical variables, historically recorded distribution patterns of indigenous malaria and the habitat of its mosquito vector Anopheles atroparvus. Overall, those individuals living in low-lying and Fenland regions exhibited higher levels of cribra orbitalia than those in nonmarshy locales. No corresponding relationship existed with enamel hypoplasia. We conclude that P. vivax malaria, in conjunction with other comorbidities, is likely to be responsible for the pattern observed. Studies of climate and infectious disease in the past are important for modeling future health in relation to climate change predictions.

Identification and functional characterization of a polymorphic oestrogen response element in the human coagulation factor IX gene promoter.

The liver expressed procoagulant factor IX (FIX) shows inter-individual variation in levels, some of which is heritable. Raised levels of FIX are associated with a thrombotic tendency. This study demonstrated that, in females but not males, part of this variation in FIX levels is due to polymorphic genotype at a locus in the factor IX gene (F9) promoter 698 bp upstream of the major transcription initiation site (-698C/T). The -698C allele (associated with higher FIX level) shows closer homology to a canonical ORE sequence and a higher binding affinity for oestrogen receptor alpha than the -698T allele. Reporter gene vectors were constructed with elements spanning residues -738 to +50 of the F9 promoter corresponding to wild type -698C and -698T alleles. A related series of vectors comprising three copies of the F9 ORE driving expression of a minimal synthetic promoter were also created. Transfection into the liver-derived HepG2 and erythroleukaemic K562 cell lines demonstrated increased levels of expression in the presence of oestrogenic factors when compared to those found in their absence; this stimulation was more pronounced in the non-liver derived K562 cell line and from the reporter vectors containing promoter elements corresponding to the -698C allele.

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

MRI staging using gadodiamide for soft-tissue tumors of the head and neck region. Results from a phase II trial and a 5-year clinical follow-up.

In order to document the safety, tolerability and efficacy of gadodiamide outside CNS, an open, non-drug comparative study was performed in patients with tumors of the head and neck region. Fifty adult patients were included and 48 patients received the contrast medium. The examinations were performed on a 1.5 T imager using transverse, non-enhanced T1- and PD-/T2-weighted conventional spin-echo sequences, followed by a contrast-enhanced transverse T1-weighted sequence. Post-contrast images provided more diagnostic information compared to unenhanced images in 33 of 48 patients (69%). This information was of significant help in four and of moderate help in 14 cases. Post-contrast images compared to non-enhanced T1-weighted showed improvement in lesion delineation for 29 of the 43 patients where a lesion was observed. Only in two patients was the diagnostic information lower post-contrast. A comparison between all pre-contrast images versus contrast medium enhanced showed post-contrast images to give more diagnostic information in 14 and less in nine patients. No patient experienced discomfort in relation to gadodiamide injection. Only one adverse event occurred which was described as thirst, being of moderate intensity. The 5-year clinical outcome was analyzed and compared with the pre-operative staging. The case-books of all patients were reviewed and in 44 patients all information could be found. Of those, 18 were still alive, one with active disease (AAD) and 17 with no evidence of disease (NED). Two of those four patients, where information was incomplete, showed NED and two had died. This trial showed that contrast-enhancement using gadodiamide for evaluation of soft tissue tumors in the head and neck region was safe and provided statistically significant more diagnostic information compared with unenhanced images. MRI, when compared with palpation/inspection, changed tumor staging in approximately 30% of all cases.

Sequence optimization in mangafodipir trisodium-enhanced liver and pancreas MRI.

To find an optimal magnetic resonance (MR) sequence for mangafodipir trisodium-enhanced liver and pancreas imaging, six healthy volunteers were studied using a 1.5 T MR system with different T1-weighted abdominal imaging sequences. These were turbo field (gradient)-echo (TFE), fast field (gradient)-echo (FFE), and spin-echo sequences before and after mangafodipir trisodium administration. Various parameter combinations were investigated within each sequence type, and then the best combination was found and compared with those of the other sequences. Signal intensity (SI) measurements were made in regions of interest in the liver, pancreas, and a reference marker with a known T1 value. Contrast index (CI, SItissue/SImarker) and contrast-to-noise ratio (CNR, [SItissue/SImarker]/SDbackground) were calculated, and percentage CI increase and CNR in the postcontrast images were used for the best sequence evaluation. Regarding CI, the TFE sequence with a TR/TE/flip angle of 15 msec/4.6 msec/20 degrees and inversion time of 300 msec had the largest pre- to postcontrast percentage increase. The FFE sequence with a TR/TE/flip angle of 140 msec/4.6 msec/90 degrees had the highest postcontrast CNR and is considered to be the optimal sequence for mangafodipir trisodium-enhanced MR imaging of the liver and pancreas.

Dynamic contrast-enhanced MRI with subtraction of aggressive soft tissue tumors after resection.

OBJECTIVE: To report the application of dynamic contrast-enhanced subtraction MRI for detecting recurrences in aggressive or malignant soft tissue tumors. DESIGN: The imaging studies consisted of static (T1- and T2-weighted spin-echo) acquisitions, followed by dynamic conventional spin-echo short TR/TE images (at 45 s, 1 min 30 s and 5 min) after a bolus of intravenous contrast medium. Contrast images were subtracted from the precontrast scan on the console. PATIENTS: Ninety-eight patients were studied who had had aggressive or malignant soft tissue tumors treated by surgery, and were followed up to assess recurrences. RESULTS: Subtraction MRI characterized recurrences better than routine sequences in 10 patients (1 lesion was seen only with this technique, 6 were better delineated, and 3 inflammatory pseudotumors were identified), and less well in 4 cases. CONCLUSION: As the number of levels studied on dynamic images is limited, and all but one recurrence were detected on T2-weighted images, it remains logical to start the examination with T2-weighted spin-echo images, and to use the dynamic study only if contrast injection is required.

MR diagnosis of head and neck tumors: comparison of contrast enhancement with triple-dose gadodiamide and standard-dose gadopentetate dimeglumine in the same patients.

OBJECTIVE: The purpose of this study was to compare the relative values of MR images obtained with a triple dose of gadodiamide and MR images obtained with the standard dose of gadopentetate dimeglumine for the detection and determination of the extent of tumors of the head and neck. MATERIALS AND METHODS: Forty-two patients with benign and malignant tumors of the head and neck were examined with contrast-enhanced MR imaging in a comparison of a triple dose of gadodiamide (0.3 mmol per kilogram body weight) and a standard dose (0.1 mmol per kilogram body weight) of gadopentetate dimeglumine. All patients underwent MR imaging with both doses of contrast material. Contrast-to-noise ratios and the percentage enhancement of normal and abnormal structures were calculated, and delineation of the lesion and the contrast between tumor and surrounding tissue were evaluated visually. RESULTS: Statistical analysis (Friedman test and Wilcoxon test) of the contrast-to-noise ratios between tumor and white matter, the percentage enhancement, and the visual assessment rating revealed a statistically significant superiority of the triple dose of gadodiamide over the standard dose of gadopentetate dimeglumine. Tumor/muscle contrast-to-noise ratios were not significantly different with the two doses. The detection rate for tumors was no better with the triple dose of gadodiamide than with the standard dose of gadopentetate dimeglumine. CONCLUSION: Use of the triple dose of gadodiamide results in a statistically significant improvement in the visual assessment rating, but it does not increase the number of tumors detected on MR images over the number detected with the standard dose of gadopentetate dimeglumine and does not appear to alter the differential diagnosis.

Action spectra of phthalocyanines with respect to photosensitization of cells.

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.

Intracellular localization of sulfonated meso-tetraphenylporphines in a human carcinoma cell line.

The intracellular localization of meso-tetraphenylporphines sulfonated to different degrees (TPPSn), in a human cervix carcinoma cell line (NHIK 3025), was studied by fluorescence microscopy and fluorescence spectroscopy. After an 18 h incubation, TPPS4, TPPS2a and TPPS2o were localized in extranuclear granules. Studies of cells stained with both TPPS4, and acridine orange, which is known to fluoresce red in lysosomes, indicated that these granules were lysosomes. In addition, a fraction of the cellbound TPPS4, TPPS2a and TPPS2o seems to be associated with the plasma membrane. Fluorescence quenching studies of cells doublestained with acridine orange and TPPS4 indicated that TPPS4 is also localized in the nucleus and in the extralysosomal cytoplasm. The intracellular location of TPPS1 differed from that of the other TPPSns studied: In 6 out of 9 experiments fluorescing extranuclear granules were found. A diffuse fluorescence extending from the perinuclear area was also observed.

Erythropoietic protoporphyria: photodynamic transfer of protoporphyrin from intact erythrocytes to other cells.

Erythrocytes in patients with erythropoietic protoporphyria (EPP) contain large amounts of protoporphyrin and are regarded as the main source of protoporphyrin in this disease. Cells in the skin of EPP patients accumulate protoporphyrin released from the erythrocytes and upon sun exposure endothelial cells are photodamaged. In the present study a light-induced transfer of protoporphyrin directly from EPP erythrocytes to cultured cells is demonstrated. Erythrocytes were layered upon cultured cells and irradiated. The nearness of erythrocyte and cultured cell membranes potentiated the transfer of protoporphyrin between these cells. This transfer was rapid and preceded the release of protoporphyrin to proteins in the medium. Further irradiation of the protoporphyrin-enriched cultured cells, after removal of the erythrocytes, caused severe photodamage to the cells and survival was dependent on both the amount of protoporphyrin transferred and on the light fluence. Clinical observations and the results of this study indicate that light energy may be involved in two steps in the pathophysiology of EPP: (A) light-induced release of protoporphyrin from erythrocytes to endothelial cells and (B) photodynamic damage to protoporphyrin-enriched endothelial cells.

Water-soluble metal naphthalocyanines--near-IR photosensitizers: cellular uptake, toxicity and photosensitizing properties in NHIK 3025 human cancer cells.

Metal naphthalocyanine complexes (MNCSs) absorb light in the near-IR spectral region (760 nm) where tissue penetration is optimal and they have been proposed as agents for photodynamic therapy (PDT). Sulphonated derivatives of tris-(2,3-naphthalocyanato) bis-chloroaluminium(III) and zinc(II) with various degrees of sulphonation were prepared. Cellular uptake, aggregation in cellular environments, cytotoxicity and photosensitizing properties were studied. Three of the four dyes studied were taken up by cells to a satisfactory degree and were not cytotoxic at the concentration used (10 micrograms ml-1). The least sulphonated sample of zinc naphthalocyanine produced some phototoxic effects (LD50 = 1.12 J cm-2). All the other samples of sulphonated naphthalocyanine were found to be aggregated inside the NHIK 3025 cells, preventing any significant PDT effect.

Intracellular localization of photosensitizers.

The intracellular localization of photosensitizers can be studied by different methods. One method involves homogenization of the cells followed by differential ultracentrifugation which leads to fractions enriched in nuclear, mitochondrial, and microsomal material as well as a supernatant fraction. More detailed information can be obtained by electron microscopy of cells exposed to light in the presence of photosensitizers. This method is based on the assumption that damage is primarily induced at intracellular sites where the concentration of photosensitizer is high. By irradiating the cells at 6 degrees C, where biochemical reactions are slow, and then incubating them for different times at 37 degrees C, it is possible to follow the development of damage. The amount of photosensitized damage to enzymes or cell functions whose localization in the cells is known gives information about the intracellular localization of the sensitizer. Fluorescence microscopy is the most direct method and is widely applicable because most photosensitizers fluoresce. Lipophilic dyes generally localize in membrane structures. In future more attention should be paid to the localization of dyes in lysosomes, as suggested by early reports. Mitochondria, the endoplasmic reticulum and nuclear membrane are other important loci for intracellular localization of sensitizers.

Hematoporphyrin ethers--III. Cellular uptake and photosensitizing properties.

1. The cellular uptake and the efficiency in sensitizing cells to photoinactivation were determined for hematoporphyrin (Hp) diphenyl ether, Hp dicyclohexyl ether and Hp dihexyl ether. 2. The phenyl diether was taken up by the cells to the same degree as was the clinically used porphyrin preparation photofrin II, while the dihexyl and notably the dicyclohexyl ether were taken up 3-4 times better. 3. Furthermore, the quantum yields for photoinactivation of cells were similar for the three diethers and twice as large as that for photofrin II. 4. Fluorescence- and absorption spectroscopy indicate that these findings are related to the fact that photofrin II is much more aggregated in the cells than are the three Hp diethers. 5. When cells loaded with the porphyrins are incubated with porphyrin-free medium containing serum a certain percentage of the cell-bound drug is removed: 14% for photofrin II, 28% for Hp diphenyl ether, 50% for Hp dicyclohexyl ether and 20% for Hp dihexyl ether. 6. With respect to cell uptake and retention of the dyes, the data did not show any uniform relationship to the polarity of the drugs, in contrast to what has been found earlier for Hp diethers of linear hydrocarbons.

Hematoporphyrin ethers--II. Improvements in method, synthesis of the dihexyl, dicyclohexanyl and diphenyl ethers and their preliminary biological evaluation.

1. Hematoporphyrin 2.4-dihexyl, -dicyclohexanyl and -diphenyl ethers have been synthesized. 2. Their chemical and physical properties are recorded. The possibility of isomerism is discussed. 3. Preliminary tests have indicated that they possess high photosensitizing efficiency on human cancer cells.