The serial millisecond crystallography instrument at the Australian Synchrotron incorporating the "Lipidico" injector.

A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an in-house developed high-viscosity injector, "Lipidico." Lipidico uses a syringe needle to extrude the microcrystal-containing viscous media and it is compatible with commercially available syringes. The combination of sample delivery via protein crystals suspended in a viscous mixture and a millisecond frame rate detector enables high-throughput serial crystallography at the Australian Synchrotron. A hit-finding algorithm, based on the principles of "robust-statistics," is employed to rapidly process the data. Here we present the first SMX experimental results with a detector frame rate of 100 Hz (10 ms exposures) and the Lipidico injector using a mixture of lysozyme microcrystals embedded in high vacuum silicon grease. Details of the experimental setup, sample injector, and data analysis pipeline are designed and developed as part of the Australian Synchrotron SMX instrument and are reviewed here.

Effect of arbuscular mycorrhizal (AM) fungi on 137Cs uptake by plants grown on different soils.

The potential use of mycorrhiza as a bioremediation agent for soils contaminated by radiocesium was evaluated in a greenhouse experiment. The uptake of (137)Cs by cucumber, perennial ryegrass, and sunflower after inoculation with a commercial arbuscular mycorrhizal (AM) product in soils contaminated with (137)Cs was investigated, with non-mycorrhizal quinoa included as a "reference" plant. The effect of cucumber and ryegrass inoculation with AM fungi on (137)Cs uptake was inconsistent. The effect of AM fungi was most pronounced in sunflower: both plant biomass and (137)Cs uptake increased on loamy sand and loamy soils. The total (137)Cs activity accumulated within AM host sunflower on loamy sand and loamy soils was 2.4 and 3.2-fold higher than in non-inoculated plants. Although the enhanced uptake of (137)Cs by quinoa plants on loamy soil inoculated by the AM fungi was observed, the infection of the fungi to the plants was not confirmed.

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells.

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Strategy for nutrient control in modern effluent treatment plants.

The fate of nutrients in the modern effluent treatment plant depends on several factors, for example type of treatment plant, availability of nutrients in the specific effluent, dosing of nutrients and sludge age/production. New technologies with the aim to increase the efficiency and stability of the conventional activated sludge process have strongly affected the possibilities to control discharge of nutrients in pulp and paper effluents. A paradox is that a reduction of organic material may often lead to an increase of nutrient discharges. It is of the utmost importance that the operators have good knowledge of the factors affecting nutrient uptake and release in order to minimise nutrient discharge and obtain optimal plant performance. Dosing of nitrogen and phosphorus is one key factor in the sensitive balance in most pulp and paper effluent treatment plants. Correct dosing is crucial as high or low doses might lead not only to increased discharge of nutrients but also to severe operational problems with poor sludge quality, which in turn affects the plant performance for longer periods.

Pig hematopoietic cell chimerism in baboons conditioned with a nonmyeloablative regimen and CD154 blockade.

BACKGROUND: In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS: Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS: In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS: These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.

Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with a porcine gammaherpesvirus.

Posttransplantation lymphoproliferative disease (PTLD) is a major complication of current clinical transplantation regimens. The lack of a reproducible large-animal model of PTLD has limited progress in understanding the pathogenesis of and in developing therapy for this clinically important disease. This study found a high incidence of PTLD in miniature swine undergoing allogeneic hematopoietic stem cell transplantation and characterized this disease in swine. Two days before allogeneic peripheral blood stem cell transplantation, miniature swine were conditioned with thymic irradiation and in vivo T-cell depletion. Animals received cyclosporine daily beginning 1 day before transplantation and continuing for 30 to 60 days. Flow cytometry and histologic examination were performed to determine the cell type involved in lymphoproliferation. Polymerase chain reaction was developed to detect and determine the level of porcine gammaherpesvirus in involved lymph node tissue. PTLD in swine is morphologically and histologically similar to that observed in human allograft recipients. Nine of 21 animals developed a B-cell lymphoproliferation involving peripheral blood (9 of 9), tonsils, and lymph nodes (7 of 9) from 21 to 48 days after transplantation. Six of 9 animals died of PTLD and 3 of 9 recovered after reduction of immunosuppression. A novel porcine gammaherpesvirus was identified in involved tissues. Miniature swine provide a genetically defined large-animal model of PTLD with many characteristics similar to human PTLD. The availability of this reproducible large-animal model of PTLD may facilitate the development and testing of diagnostic and therapeutic approaches for prevention or treatment of PTLD in the clinical setting.

Identification of novel porcine endogenous betaretrovirus sequences in miniature swine.

PCR amplification of genomic DNA from miniature swine peripheral blood lymphocytes, using primers corresponding to highly conserved regions of the polymerase (pol) gene, allowed the identification of two novel porcine endogenous retrovirus (PERV) sequences, PMSN-1 and PMSN-4. Phylogenetic analyses of the nucleotide sequences of PMSN-1 and PMSN-4 revealed them to be most closely related to betaretroviruses. The identification of PERVs belonging to the Betaretrovirus genus shows that endogenous retroviruses of this family are more broadly represented in mammalian species than previously appreciated. Both sequences contained inactivating mutations, implying that these particular loci are defective. However, Southern blot analysis showed additional copies of closely related proviruses in the miniature swine genome. Analyses of fetal and adult miniature swine tissues revealed a broad mRNA expression pattern of both PMSN-1 and PMSN-4. The most abundant expression was detected in whole bone marrow c-kit(+) (CD117(+)) progenitor bone marrow cells, fetal liver, salivary gland, and thymus. It appears unlikely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The betaretrovirus sequences described in this report will allow such investigations to be actively pursued.

Clonal immunoglobulin light chain variable region germline gene use in AL amyloidosis: association with dominant amyloid-related organ involvement and survival after stem cell transplantation.

AL (primary or immunoglobulin light chain) amyloidosis (AL) differs from myeloma per se in that tissue deposits of amyloid are found, typically in association with small numbers of clonal plasma cells producing lambda light chains, and also in that AL patients typically present with a predominantly dysfunctional organ-system. This constellation of features - fibrillar deposits comprised of light chains, lambda light chain predominance, and organ-system tropism and dysfunction - remains unexplained. Select patients with AL respond to haemopoietic stem cell transplantation (SCT) with clinical improvement and extended survival, particularly those who do not have cardiac involvement. In order to ascertain whether the organ-system tropism of AL was associated with immunoglublin light chain variable region (Ig VL) germline gene utilization, we attempted to clone, sequence and assign germline donors to the clonal Ig VL genes of 62 AL patients, all of whom were treated with SCT. We succeeded in 39 cases, identifying clonal AL genes derived from donors of the lambdaI (n = 10), lambdaII (n = 5), lambdaIII (n = 6), lambdaVI (n = 11) and KI (n = 7) subtypes. The majority of the donors (IGLV6S1, DPL5, DPL2, DPL23 and LFVK431) were genes that appear in the expressed repertoire <5% of the time, suggesting an intrinsic propensity to form amyloid under certain conditions. Patients whose clones derived from the lambdaVI IGLV6S1 donor uniformly presented with dominant renal involvement while those with other Vlambda or unknown donors often had dominant cardiac or other organ involvement, particularly patients whose clones derived from the lambdaI DPL2 donor. In addition, both early (<3 months) and overall post-SCT survival were significantly better in lambdaVI IGLV6S1 patients compared to patients with other Vlambda donors. These findings indicate that there are important associations in AL amyloidosis among Ig VL gene utilization, organ-system tropism and post-SCT survival.

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.

A simple screening test for variant transthyretins associated with familial transthyretin amyloidosis using isoelectric focusing.

Variant forms of the plasma protein transthyretin (TTR) are associated with the most frequently occurring type of familial systemic amyloidosis. Organ system involvement in transthyretin type amyloidosis (ATTR) is often similar to that which occurs in light chain amyloid disease (AL). The proper diagnosis of ATTR is important since treatment (liver transplantation) differs from that in AL (chemotherapy). We present a two-step test to screen sera for variant TTRs using non-denaturing gel electrophoresis performed in 7.5% acrylamide (PAGE) followed by isoelectric focusing (IEF) between pH 4.0 and 7.0 in 2.5 M urea. Serum samples from 110 patients with amyloidosis and their relatives were tested using this IEF technique and compared to genetic mutation results. Sera from patients with ATTR who underwent liver transplantation were also examined prior to and following surgery. IEF analysis showed the presence of both wild-type and variant TTR in 74 of the 110 serum samples tested. Genomic DNA from peripheral blood was used to identify TTR gene mutations in 77 of the 110 patients. Fifteen variants including Val122Ile, preponderant in the African-American population, could be demonstrated by IEF. The sensitivity of IEF was 96% (74/77) and the specificity was 100% (33/33). The predictive values for a positive or negative result were 100% (74/74) and 92% (33/36), respectively. There were no false-positive results and 4% (3/77) false-negative results. In sera from patients with ATTR who underwent liver transplantation, variant TTR was detected by IEF before, but not after, surgery. A simple, accurate, sensitive method is presented as a useful screening test for variant transthyretins associated with ATTR.

Chemical characterization and reactivity of iron chelator-treated amphibole asbestos.

Iron in amphibole asbestos is implicated in the pathogenicity of inhaled fibers. Evidence includes the observation that iron chelators can suppress fiber-induced tissue damage. This is believed to occur via the diminished production of fiber-associated reactive oxygen species. The purpose of this study was to explore possible mechanisms for the reduction of fiber toxicity by iron chelator treatments. We studied changes in the amount and the oxidation states of bulk and surface iron in crocidolite and amosite asbestos that were treated with iron-chelating desferrioxamine, ferrozine, sodium ascorbate, and phosphate buffer solutions. The results have been compared with the ability of the fibers to produce free radicals and decompose hydrogen peroxide in a cell-free system in vitro. We found that chelators can affect the amount of iron at the surface of the asbestos fibers and its valence, and that they can modify the chemical reactivity of these surfaces. However, we found no obvious or direct correlations between fiber reactivity and the amount of iron removed, the amount of iron at the fiber surface, or the oxidation state of surface iron. Our results suggest that surface Fe3+ ions may play a role in fiber-related carboxylate radical formation, and that desferrioxamine and phosphate groups detected at treated fiber surfaces may play a role in diminishing and enhancing, respectively, fiber redox activity. It is proposed that iron mobility in the silicate structure may play a larger role in the chemical reactivity of asbestos than previously assumed.

Plant nutrition, dry matter gain and partitioning at the whole-plant level.

Differential flows of photoassimilate result in patterns of dry matter distribution among plant organs. The extent to which these patterns are dependent on the flux of different mineral nutrients entering the root and the extent to which the distribution of dry matter in the whole plant is affected by differentials in nutrient (primarily nitrate) flux among parts of the root system is considered. It is concluded that patterns of dry matter distribution and nutritional status may depend on how nutrient supply has been manipulated about the root. Where the flux density of nutrient has been decreased and has become limiting to plant growth, two categories of response have been observed. In the case of N, P or S, limiting flux density results in a proportionately greater amount of plant dry matter in roots than is found at higher flux densities. This contrasts with the case of limiting K, Mg or Mn supply, where proportionately less plant dry matter is found in roots at lower nutrient flux densities than at higher flux densities. In the case of N, particular attention is paid as to how sink strength may be related to differences between root and leaf cells in their capacity for loosening and synthesis processes in the primary cell wall.

Effects of the antihypertensive drug captopril on human salivary secretion rate and composition.

The effects of the antihypertensive drug captopril on salivary secretion rate and composition was evaluated in 24 healthy adults (18-46 yr) according to a double-blind, cross-over design. Unstimulated and paraffin-chewing stimulated whole saliva and 3% citric acid stimulated parotid and submandibular-sublingual (SM-SL) secretion were collected at 10.30 a.m. (about 2h after intake of breakfast) on day 0 (baseline values), day 1 (experimental acute values) and day 7 (experimental chronic values) in each treatment period. In 8 of the subjects, also morning samples were collected at 7.30 a.m., with the test subjects in a fasting condition. Whole saliva was assessed for flow rate and for concentrations of sodium, potassium, chloride, calcium and phosphate. In addition, parotid and SM-SL secretion were assessed for concentrations of total protein, hexosamine, sialic acid, lactoferrin and salivary IgA and for activities of amylase, lysozyme and salivary peroxidase. During treatment with the angiotensin converting enzyme inhibitor captopril, the secretion rates tended to increase for unstimulated and paraffin-chewing stimulated whole saliva and for parotid secretion. For salivary composition, no alterations were observed in any of the collected secretions.

Allocation of mass in trees subject to nitrogen and magnesium limitation.

It has previously been shown that lack of magnesium, potassium or manganese depresses allocation of new mass to roots in trees, and that these elements often occur in low amounts in declining forest stands. A model for tree growth and nutrient uptake was used to examine the effect of allocation pattern of new plant mass on growth and development in soils with low nutrient concentrations. Growth parameters and empirical allocation functions from steady-state experiments with birch (Betula pendula Roth.) and Norway spruce (Picea abies (L.) Karst.) limited by N or Mg were used in the simulations. The simulation results show that plant nutrient concentration is sensitive to decreases in soil Mg concentration and is not counteracted by an increased allocation of plant mass to roots. A 50% reduction in the optimal soil Mg concentration decreased the plant Mg concentration to the threshold of survival, whereas a 50% reduction of soil N had much less effect. Birch was more sensitive to low soil Mg concentration than Norway spruce. We conclude that, in the discussion of forest decline, it may be important to consider the decreased allocation of mass to roots when Mg (or K or Mn) is in short supply.

Growth and nutrition of birch seedlings at varied relative addition rates of magnesium.

Growth and nutrition of hydroponically cultivated birch seedlings (Betula pendula Roth.) were investigated at various magnesium (Mg) availabilities. Suboptimum Mg conditions were created by adding Mg once per hour in exponentially increasing amounts at one of four relative addition rates (R(Mg)): 0.05, 0.10, 0.15 or 0.20 day(-1). Seedlings given free access to Mg were used as controls. After an acclimation period, the relative growth rate of the seedlings attained the same value as the corresponding relative rate of Mg addition. In all suboptimum Mg treatments, deficiency symptoms in the form of chloroses and necroses developed in the older leaves, both during and after the phase of growth acclimation. The severity of these symptoms was correlated with the availability of Mg. The relative growth rate of seedlings was linearly correlated with plant Mg status. The root fraction of the total biomass decreased from 22% in control plants to 8% in plants receiving the lowest rate of Mg addition. A shift in Mg availability from free access to R(Mg) = 0.05 day(-1) decreased the photosynthetically active leaf area per plant weight, despite a concomitant increase in the leaf weight ratio (leaf dry weight/plant dry weight) from 0.61 to 0.75. The loss in assimilating leaf area was mainly a consequence of enhanced leaf mortality and formation of necroses, and to a minor extent attributable to increased carbon costs for leaf area production. A decrease in starch concentration was observed in leaves showing Mg-deficiency symptoms, whereas the starch concentration in healthy leaves was unaffected by Mg availability. It was concluded that shortage of carbohydrates constituted the major growth constraint, particularly for roots, under Mg-limiting conditions.

Effects of the beta-adrenoceptor antagonists atenolol and propranolol on human parotid and submandibular-sublingual salivary secretion.

The aim of the study was to evaluate the effects of a beta 1-selective (atenolol 50 mg q.d.) and a non-selective (propranolol 80 mg b.i.d.) beta-adrenoceptor antagonists on human stimulated parotid and submandibular-sublingual (SM-SL) gland secretion. A randomized double-blind, placebo-controlled cross-over ("Latin square") design was used in 19 healthy male volunteers. Stimulated parotid and SM-SL saliva were sampled immediately before and 7 days after the start of each treatment period. Stimulation of salivary secretion was achieved by use of a 3% citric acid solution. Plasma concentrations of propranolol and atenolol were determined from blood samples. The salivary secretion of both glands was assessed for flow rate, amylase, lysozyme, and salivary peroxidase activity and for concentrations of total protein, hexosamine, sialic acid, Ca2+, Cl-, K+, Mg2+, Na+, and PO4(3-). In both parotid and SM-SL secretions, the total protein and phosphate concentrations and amylase activity were significantly decreased during the two active treatment periods. In SM-SL gland secretion, there were significant changes in potassium and calcium concentrations during active treatment as compared with baseline, with potassium showing a decreased and calcium an increased concentration. During atenolol treatment, salivary peroxidase activity decreased significantly in SM-SL secretion. In parotid secretion, the hexosamine/total protein ratio decreased and the sialic acid/hexosamine ratio increased during atenolol treatment, which may indicate an effect on protein synthesis. No significant effects on salivary secretion rates were disclosed.