Development and comparison of the methods for quantitative electron probe X-ray microanalysis analysis of thin specimens and their application to biological material.

In recent years, there has been a return to the use of electron probe X-ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff-Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff-Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin-embedded materials emphasized.

Loss of CDX2 and high COX2 (PTGS2) expression in metastatic colorectal cancer.

Lack of expression of the tumour suppressor gene caudal-type homeobox 2 (CDX2) associates with poor outcomes in early stage colorectal cancer (CRC). Yet its prognostic value in the context of other prognostic biomarkers in metastatic CRC (mCRC) is unknown. Overexpressed cyclooxygenase-2 (COX2) has been reported in advanced CRC. However, CDX2 and COX2 relationship in mCRC remains undetermined. We aimed to assess their expression in mCRC tumours from a clinically characterised cohort and their influence on overall survival (OS) and progression-free survival (PFS) in first line. Among 720 consecutive mCRC patients, 346 had tumour samples appropriate for tissue microarray assembly and immunohistochemistry analyses. Clinical and survival data were retrospectively assessed. Loss of CDX2 expression was detected in 27 (7.8%) samples, enriched in poorly differentiated tumours (20%; p < 0.01) and in those with the BRAF p.V600E variant (40%; p < 0.01). Most tumours (93.4%) expressed COX2. COX2-negative samples were enriched in poorly differentiated mCRC. In unadjusted analyses, median OS (p < 0.001) and median PFS (p < 0.05) were inferior for patients with CDX2-negative versus CDX2-positive tumours. In conclusion, loss of CDX2 was significantly associated with poorly differentiated mCRC and BRAF p.V600E allele and a prognostic marker of worse OS.

Lysosomal responses to different gold forms (nanoparticles, aqueous, bulk) in mussel digestive cells: a trade-off between the toxicity of the capping agent and form, size and exposure concentration.

Gold nanoparticles (NPs) are increasingly used in technological materials and consumer products and may have toxicological characteristics distinct from bulk and aqueous gold. The aim of this work was to understand the effects of Au NPs especially, how the form, the size and the coating influence bioaccumulation/biodistribution and toxicity of NPs in mussels, Mytilus galloprovincialis. Mussels were exposed for 3 d to concentrations of Au (0.75, 75 and 750 µg Au/l) supplied as Au-Cit NPs (5 and 40 nm; Au5-Cit and Au40-Cit), bulk and aqueous Au (HAu(III)Cl4), and to the capping agent (Na-citrate) in doses used in the formulation of NPs (0.005, 0.5, 5 mg/l). Citrate-stabilised NPs formed stable suspensions of aggregates in seawater (SW) available for mussels. Au accumulation in soft tissues was similar in Au40-Cit and aqueous Au exposed mussels, lower in Au5-Cit and negligible after bulk exposure. Au NPs were identified (X-ray microanalysis) in different compartments of the endolysosomal system in digestive cells, and small size NPs (5 nm) were more accumulated than 40 nm NPs, aqueous and bulk. The degree of lysosomal membrane destabilisation was related with intralysosomal metal accumulation and depended on the form, NP size (Au5-Cit > Au40-Cit > aqueous > bulk) and concentration. Citrate alone provoked extreme reduction in lysosomal membrane stability. Toxicopathic alterations were recorded in digestive gland cells (vacuolisation, swollen RER, connective tissue disruption and cell death) especially in mussels exposed to 40 nm NPs. Deleterious effects resulted from digestive tract obliteration (agglomerates) and digestion malfunction. The toxic effect of Au-Cit NPs was influenced both by NP size, capping agent composition and the dose of capping agent carried by NPs, which was size dependent.

Electron beam damage studies of synthetic 6-line ferrihydrite and ferritin molecule cores within a human liver biopsy.

In order to achieve an accurate understanding of the crystal structure of 6-line ferrihydrite (6LFh) and ferritin molecule cores within a human liver biopsy using transmission electron microscopy (TEM), electron beam damage should be considered. For the case of 6LFh, the electron energy loss near-edge structure (ELNES) of core ionisation edges in the electron energy loss spectrum (EELS) combined with multiple linear least-square (MLLS) fitting of reference spectra together with analysis of selected area electron diffraction (SAED) patterns suggests that the iron in 6LFh is solely octahedrally coordinated Fe3+. With increasing electron dose, an increasing percentage of this octahedrally coordinated Fe3+ migrates to tetrahedral sites. When the dose exceeds 3 x 10(8) electrons/nm2, Fe2+ is found to be present in the material. This method also indicates that the iron in ferritin molecule cores within a human liver biopsy is the same as in 6LFh, entirely Fe3+ in octahedral coordination with oxygen. Again the percentage of octahedrally coordinated Fe3+ decreases as the accumulated electron dose increases and Fe2+ is produced in the liver biopsies when the electron dose exceeds 10(6)electrons/nm2.

Chronic administration of sodium cyanate decreases O2 extraction ratio in dogs.

It has been proposed that an increase in the affinity of hemoglobin for O2 may be beneficial in severe hypoxemia. To test this hypothesis, we compared the response to progressive hypoxemia in dogs with normal hemoglobin affinity (P50 = 32.4 +/- 0.7 Torr) to dogs with a left shift of the oxyhemoglobin dissociation curve (P50 = 21.9 +/- 0.5 Torr) induced by chronic oral administration of sodium cyanate. Animals were anesthetized, paralyzed, and mechanically ventilated. The inspired O2 fraction was progressively lowered by increasing the inspired fraction of N2. The lowest level of O2 transport required to maintain base-line O2 consumption (VO2) was 9.3 +/- 0.8 ml.min-1.kg-1 for control and 16.5 +/- 1.1 ml.min-1.kg-1 for the sodium cyanate-treated dogs (P less than 0.01). Other measured parameters at this level of O2 transport were, for experimental vs. control: arterial PO2 19.3 +/- 2.4 (SE) Torr vs. 21.8 +/- 1.6 Torr (NS); arterial O2 content 10.0 +/- 1.2 ml/dl vs. 4.9 +/- 0.4 ml/dl (P less than 0.01); mixed venous PO2 14.0 +/- 1.5 Torr vs. 13.8 +/- 1.0 Torr (NS); mixed venous O2 content 6.8 +/- 1.0 ml/dl vs. 2.3 +/- 0.2 ml/dl (P less than 0.01); and O2 extraction ratio 32.7 +/- 2.8% vs. 51.2 +/- 3.8% (P less than 0.01). We conclude that chronic administration of sodium cyanate appears to be detrimental to O2 transport, since the experimental dogs were unable to increase their O2 extraction ratios to the same level as control, thus requiring a higher level of O2 transport to maintain their base-line VO2 values.

Oxygen delivery and utilization in hypothermic dogs.

Hypothermia produces a decrease in metabolic rate that may be beneficial under conditions of reduced O2 delivery (Do2). Another effect of hypothermia is to increase the affinity of hemoglobin for O2, which can adversely affect the release of O2 to the tissues. To determine the overall effect of hypothermia on the ability of the peripheral tissues to extract O2 from blood, we compared the response to hypoxemia of hypothermic dogs (n = 8) and of normothermic controls (n = 8). The animals were anesthetized, mechanically ventilated, and paralyzed to prevent shivering. The inspired concentration of O2 was progressively reduced until the dogs died. The core temperatures of the control and hypothermic dogs were 37.7 +/- 0.3 and 30.5 +/- 0.1 degree C, respectively (P less than 0.01). The O2 consumption (VO2) of the control dogs was significantly greater than that of the hypothermic dogs (P less than 0.05), being 4.7 +/- 0.4 and 3.2 +/- 0.3 ml X min-1 X kg-1, respectively. Hypothermia produced a left shift of the oxyhemoglobin dissociation curve (ODC) to a PO2 at which hemoglobin is half-saturated with O2 of 19.8 +/- 0.7 Torr (control = 32.4 +/- 0.7 Torr, P less than 0.01). The O2 delivery at which the VO2 becomes supply dependent (DO2crit) was 8.5 ml X min-1 X kg-1 for control and 6.2 ml X min-1 X kg-1 for hypothermia. The hypothermic dogs maintained their base-line VO2's at lower arterial PO2's than control.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of nebulized salbutamol therapy on the incidence of postoperative chest infection in high risk patients.

Patients who smoke heavily and those with pre-existing airflow obstruction are at particular risk of postoperative respiratory infection following upper abdominal surgery. This invariably prolongs hospital stay and increases morbidity. In order to determine whether high dose bronchodilator therapy in the perioperative period reduced the risk of infection, all patients undergoing elective upper abdominal surgery were assessed for risk of developing postoperative infection. Fifty-three patients were identified as high risk according to previously published criteria and were randomly allocated to receive nebulized salbutamol (5 mg) or saline placebo 6 hourly for 48 h beginning 1 h preoperatively. There was no difference in rates of postoperative chest infection in the two groups and this study, therefore, provides no support for the routine preoperative use of bronchodilators in these patients.

Tuberculosis and HIV seroprevalence in Lambeth, Southwark and Lewisham, an area of South London.

Since the mid-1980s the number of cases of TB notified within the U.K. has continued to rise although the contribution of HIV to this rise remains unclear. A 12-month prospective cohort study was conducted at chest and HIV clinics in four hospitals in Lambeth, Southwark and Lewisham (LSL), an area of South London, to determine the proportion of patients with culture-proven TB infected with HIV. Secondary aims were to determine the proportion of patients with TB and undiagnosed HIV at first presentation to chest clinics, to determine the proportion of patients presenting with TB as an AIDS defining illness (ADI) and to identify risk factors for co-infection with TB and HIV. In chest clinics, demographic data and left-over blood from patients aged 16 or over with culture-proven TB was collected, anonymised and HIV tested. In HIV clinics, demographic data on patients with TB already known to be HIV seropositive were also obtained. Twenty-one patients (13%, 95% CI-8-19%) of 159 with culture-proven TB were infected with HIV Four (3%) of 133 patients at first presentation to chest clinics had undiagnosed HIV; two were subsequently diagnosed. Of the 21 patients withTB and HIV, nine (43%) presented with TB as an ADI. Patients with TB and HIV were significantly more likely to be aged between 35 and 55 years compared to HIV seronegative patients [12/21 (57%) vs. 38/138 (28%), P=0.006]. None of the patients from the Indian Subcontinent were HIV seropositive [0/21 vs. 25/138 (18%), P=0.047]. At the present time, universal HIV testing of patients with culture-provenTB in chest clinics within the U.K. is unlikely to significantly reduce the number of patients with undiagnosed HIV.

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells.

Decreases in the intracellular concentrations of both K(+) and Cl(-) have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied.

Immunolocalization of duodenal cytochrome B: a relationship with circulating markers of iron status.

BACKGROUND: The brush border ferric reductase (Dcytb) is critical for the absorption of dietary iron and appears to be expressed on the duodenal enterocyte brush border. The Dcytb expression is increased in severe iron-deficient anaemia, but the situation in a more typical mild iron deficiency is unclear. This study investigated Dcytb expression in patients with normal iron status or mild iron deficiency and its relationships with enterocyte iron status. MATERIALS AND METHODS: Duodenal biopsy specimens and blood samples were obtained from 32 patients undergoing routine upper gastrointestinal endoscopy. Twenty-three specimens (six iron-deficient and 17 iron-replete) were processed for light-microscopy (LM) and for immunohistochemistry with antibodies against Dcytb and heavy/light chain ferritin subunits. The nine remaining biopsies (three iron-deficient and six iron-replete) were processed for electron microscopy (EM). Immunolocalization of Dcytb and intracellular ferritin was performed with appropriate primary antibodies followed by 10-nm gold conjugate labels. RESULTS: The LM process showed a strong negative correlation between immunolabelling intensity of Dcytb on the enterocyte brush border and serum iron saturation (P < 0.001), but only a weak negative correlation between this antigen and haemoglobin (P = 0.08) or serum ferritin concentrations (P = 0.4). EM confirmed anti-Dcytb preferential labelling of microvilli rather than enterocyte cytoplasm (P = 0.001), but preferential antiferritin labelling of cytoplasm (P < 0.02). There was no correlation with enterocyte cytoplasmic ferritin labelling (i.e. enterocyte iron status and Dcytb expression). CONCLUSIONS: Enterocyte Dcytb brush border expression is increased even in mild iron deficiency and may be related to serum iron saturation. The lack of correlation with enterocyte ferritin expression deserves further study with direct measurement of intracellular iron.

Biphasic behavior of changes in elemental composition during staurosporine-induced apoptosis.

Although the identification of events that occur during apoptosis is a fundamental goal of apoptotic cell death research, little is know about the precise sequence of changes in total elemental composition during apoptosis. We evaluated total elemental composition (Na, Mg, P, Cl, S, and K) in relation to molecular and morphological features in human U937 cells induced to undergo apoptosis with staurosporine, an intrinsic pathway activator. To evaluate total elemental content we used electron probe X-ray microanalysis to measure simultaneously all elements from single, individual cells. We observed two phases in the changes in elemental composition (mainly Na, Cl and K). The early phase was characterized by a decrease in intracellular K (P<0.001) and Cl (P<0.001) content concomitant with cell shrinkage, and preceded the increase in proteolytic activity associated with the activation of caspase-3. The later phase started with caspase-3 activation, and was characterized by a decrease in the K/Na ratio (P<0.001) as a consequence of a significant decrease in K and increase in Na content. The inversion of intracellular K and Na content was related with the inhibition of Na+/K+ ATPase. This later phase was also characterized by a significant increase (P<0.001) in intracellular Cl with respect to the early phase. In addition, we found a decrease in S content and an increase in the P/S ratio. These distinctive changes coincided with chromatin condensation and DNA fragmentation. Together, these findings support the concept that changes in total elemental composition take place in two phases related with molecular and morphological features during staurosporine-induced apoptosis.

The preparation of cultured cells for X-ray microanalysis.

X-ray microanalysis of cells in culture is being used increasingly for the study of relationships between element (ion) content and cell function. There is, however, no one single method which can be used for the preparation of all different cell types for study by microanalysis. Cells in suspension are usually concentrated by centrifugation, before cryofixation, cryosectioning, and freeze drying. On the other hand cells grown as monolayers are more often studied as whole cell mounts, which are washed to remove the external medium before cryofixation and freeze drying. The alternative approach, sectioning of cell monolayers is rarely used. Some of the difficulties encountered in preparing and monolayers of smooth muscle cells for X-ray microanalysis are discussed here.

Changes in sodium concentration in cardiac myocytes from diabetic rats.

The effects of streptozotocin induced diabetes on rats were studied. The animals showed an increase in blood glucose concentration and a loss of weight from both the body and the heart. Loss of weight from the heart was less severe leading to an increased heart to body weight ratio. Study of element concentrations by X-ray microanalysis showed that there was an increase in intracellular Na concentration in cardiac myocytes from the diabetic animals, but no change in Mg. These results agree with studies which show changes in Na/K ATPase after the onset of diabetes.

Concentrations of elements in rat thymocytes measured by X-ray microanalysis.

Elemental concentrations of rat thymocytes in vivo were studied by X-ray microanalysis of freeze-dried sections. Cells from different regions, the subcapsular zone, the cortex and the medulla were studied in thymic tissue from a number of animals. Generally thymocytes situated in the medulla had higher concentrations of K compared to those in the subcapsular zone. The concentration of Na in the nucleus was constant in the medulla in all animals but some variation in this element was seen between animals in the subcapsular zone. The distribution of K/Na ratio in individual thymocytes was different in each region of the thymus. Cells with low K/Na ratio (less than 5) were predominant in the subcapsular zone, whereas cells with higher values for K/Na ratio were found in the cortex and medulla. The subcapsular zone is the region where mitotic cells are mostly situated. The finding of thymocytes with higher concentrations of Na and low K/Na ratios in this region is in accord with in vitro studies on thymocyte stimulation.

A comparison of the elemental concentrations in tissue thymocytes from diabetic and non-diabetic rats.

The elemental concentrations in thymocytes from control and diabetic rats were studied by use of the techniques of cryofixation and X-ray microanalysis to determine whether any changes occur in the diseased state. Decreases in the concentrations of the elements P and K were found in thymocytes from the subcapsular and cortical regions of the gland in thymus tissue taken 4 days after the onset of diabetes. A decrease in the concentration of Mg was also found in thymocytes from the subcapsular region of the gland. These changes suggest that the metabolism of thymocytes in vivo is altered in the diabetic animals.

Elemental concentrations in isolated rat thymocytes prepared for cryofixation in the presence of different media.

Rat thymocytes were isolated in suspension and the effect of preparing the cells for cryofixation in the presence of different media on the elemental content was investigated using the technique of X-ray microanalysis. Cells prepared in the different media showed variation in the concentrations of Na, K and Cl. The isolated cells were incubated at 310 K for 1 h to allow recovery from isolation. There was a decrease in Na and Cl content after incubation. The thymocyte population was disturbed by suspension in medium containing dextran, and this resulted in a number of cells with high concentrations of Na and low concentrations of K. These cells did not take up vital dye. Thymocytes were also prepared for freezing by using high-speed centrifugation to concentrate the cells. Thymocytes prepared by this method showed values for concentrations of Na, K and Cl similar to published values for these cells using other methods of estimation. There were, however, consistent differences in Na content between the cells prepared in Hanks' balanced salt solution and those prepared in serum. Factors which affect the apparent concentration of Na and Cl in isolated cells are discussed.

X-ray microanalysis of cells in suspension and the application of this technique to the study of the thymus gland.

Although X-ray microanalysis has become a well established technique in the study of tissues there are still relatively few papers reporting results from cells in culture. Our early investigations on freshly isolated murine thymocytes reported higher than expected concentrations of Na and Cl in these cells but subsequent studies have shown that these high concentrations are artefactual. Rat thymocytes that have been isolated and incubated for a short time to allow them to recover from the isolation procedure have lower concentrations of Na and Cl. Sections prepared from pellets of cells which have been concentrated by centrifugation in a microhaematocrit centrifuge have concentrations of Na and Cl which are within the range that has been described for thymocytes in tissues. This method of concentration can be applied successfully to the study of white cells from the peripheral blood. In diabetic animals pyknotic cells occur in the thymus. These cells are characterised by low concentrations of K but no rise in the concentration of Na. Study of thymocytes isolated from a diabetic animal also show pyknotic cells low in K confirming these observations.