Single-cell RNA-sequencing of virus-specific cellular immune responses in chronic hepatitis B patients.

Chronic hepatitis B (CHB) is a major global health challenge. CHB can be controlled by antivirals but a therapeutic cure is lacking. CHB is characterized by limited HBV-specific T cell reactivity and functionality and expression of inhibitory receptors. The mechanisms driving these T cell phenotypes are only partially understood. Here, we created a single-cell RNA-sequencing dataset of HBV immune responses in patients to contribute to a better understanding of the dysregulated immunity. Blood samples of a well-defined cohort of 21 CHB and 10 healthy controls, including a subset of 5 matched liver biopsies, were collected. scRNA-seq data of total immune cells (55,825) plus sorted HBV-specific (1,963), non-naive (32,773) and PD1+ T cells (96,631) was generated using the 10X Genomics platform (186,123 cells) or the full-length Smart-seq2 protocol (1,069 cells). The shared transcript count matrices of single-cells serve as a valuable resource describing transcriptional changes underlying dysfunctional HBV-related T cell responses in blood and liver tissue and offers the opportunity to identify targets or biomarkers for HBV-related immune exhaustion.

Structural basis for dimerization of a paramyxovirus polymerase complex.

The transcription and replication processes of non-segmented, negative-strand RNA viruses (nsNSVs) are catalyzed by a multi-functional polymerase complex composed of the large protein (L) and a cofactor protein, such as phosphoprotein (P). Previous studies have shown that the nsNSV polymerase can adopt a dimeric form, however, the structure of the dimer and its function are poorly understood. Here we determine a 2.7 Å cryo-EM structure of human parainfluenza virus type 3 (hPIV3) L-P complex with the connector domain (CD') of a second L built, while reconstruction of the rest of the second L-P obtains a low-resolution map of the ring-like L core region. This study reveals detailed atomic features of nsNSV polymerase active site and distinct conformation of hPIV3 L with a unique ß-strand latch. Furthermore, we report the structural basis of L-L dimerization, with CD' located at the putative template entry of the adjoining L. Disruption of the L-L interface causes a defect in RNA replication that can be overcome by complementation, demonstrating that L dimerization is necessary for hPIV3 genome replication. These findings provide further insight into how nsNSV polymerases perform their functions, and suggest a new avenue for rational drug design.

Nucleolin binds to and regulates transcription of hepatitis B virus covalently closed circular DNA minichromosome.

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.

Proteomics of immune cells from liver tumors reveals immunotherapy targets.

Elucidating the mechanisms by which immune cells become dysfunctional in tumors is critical to developing next-generation immunotherapies. We profiled proteomes of cancer tissue as well as monocyte/macrophages, CD4+ and CD8+ T cells, and NK cells isolated from tumors, liver, and blood of 48 patients with hepatocellular carcinoma. We found that tumor macrophages induce the sphingosine-1-phospate-degrading enzyme SGPL1, which dampened their inflammatory phenotype and anti-tumor function in vivo. We further discovered that the signaling scaffold protein AFAP1L2, typically only found in activated NK cells, is also upregulated in chronically stimulated CD8+ T cells in tumors. Ablation of AFAP1L2 in CD8+ T cells increased their viability upon repeated stimulation and enhanced their anti-tumor activity synergistically with PD-L1 blockade in mouse models. Our data reveal new targets for immunotherapy and provide a resource on immune cell proteomes in liver cancer.

T-cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma.

BACKGROUND & AIMS: Despite recent translation of immunotherapies into clinical practice, the immunobiology of hepatocellular carcinoma (HCC), in particular the role and clinical relevance of exhausted and liver-resident T cells remain unclear. We therefore dissected the landscape of exhausted and resident T cell responses in the peripheral blood and tumor microenvironment of patients with HCC. METHODS: Lymphocytes were isolated from the blood, tumor and tumor-surrounding liver tissue of patients with HCC (n = 40, n = 10 treated with anti-PD-1 therapy). Phenotype, function and response to anti-PD-1 were analyzed by mass and flow cytometry ex vivo and in vitro, tissue residence was further assessed by immunohistochemistry and imaging mass cytometry. Gene signatures were analyzed in silico. RESULTS: We identified significant enrichment of heterogeneous populations of exhausted CD8+ T cells (TEX) in the tumor microenvironment. Strong enrichment of severely exhausted CD8 T cells expressing multiple immune checkpoints in addition to PD-1 was linked to poor progression-free and overall survival. In contrast, PD-1 was also expressed on a subset of more functional and metabolically active CD103+ tissue-resident memory T cells (TRM) that expressed few additional immune checkpoints and were associated with better survival. TEX enrichment was independent of BCLC stage, alpha-fetoprotein levels or age as a variable for progression-free survival in our cohort. These findings were in line with in silico gene signature analysis of HCC tumor transcriptomes from The Cancer Genome Atlas. A higher baseline TRM/TEX ratio was associated with disease control in anti-PD-1-treated patients. CONCLUSION: Our data provide information on the role of peripheral and intratumoral TEX-TRM dynamics in determining outcomes in patients with HCC. The dynamics between exhausted and liver-resident T cells have implications for immune-based diagnostics, rational patient selection and monitoring during HCC immunotherapies. LAY SUMMARY: The role of the immune response in hepatocellular carcinoma (HCC) remains unclear. T cells can mediate protection against tumor cells but are frequently dysfunctional and exhausted in cancer. We found that patients with a predominance of exhausted CD8+ T cells (TEX) had poor survival compared to patients with a predominance of tissue-resident memory T cells (TRM). This correlated with the molecular profile, metabolic and functional status of these cell populations. The enrichment of TEX was independently associated with prognosis in addition to disease stage, age and tumor markers. A high TRM proportion was also associated with better outcomes following checkpoint therapy. Thus, these T-cell populations are novel biomarkers with relevance in HCC.

Short-baseline interferometry local-tie experiments at the Onsala Space Observatory.

We present results from observation, correlation and analysis of interferometric measurements between the three geodetic very long baseline interferometry (VLBI) stations at the Onsala Space Observatory. In total, 25 sessions were observed in 2019 and 2020, most of them 24 h long, all using X band only. These involved the legacy VLBI station ONSALA60 and the Onsala twin telescopes, ONSA13NE and ONSA13SW, two broadband stations for the next-generation geodetic VLBI global observing system (VGOS). We used two analysis packages: ν Solve to pre-process the data and solve ambiguities, and ASCOT to solve for station positions, including modelling gravitational deformation of the radio telescopes and other significant effects. We obtained weighted root mean square post-fit residuals for each session on the order of 10-15 ps using group-delays and 2-5 ps using phase-delays. The best performance was achieved on the (rather short) baseline between the VGOS stations. As the main result of this work, we determined the coordinates of the Onsala twin telescopes in VTRF2020b with sub-millimetre precision. This new set of coordinates should be used from now on for scheduling, correlation, as a priori for data analyses, and for comparison with classical local-tie techniques. Finally, we find that positions estimated from phase-delays are offset ∼ + 3  mm in the up-component with respect to group-delays. Additional modelling of (elevation dependent) effects may contribute to the future understanding of this offset.

De Novo Emergence of Peptides That Confer Antibiotic Resistance.

The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes.IMPORTANCEDe novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

Impact of the terrestrial reference frame on the determination of the celestial reference frame.

Currently three up-to-date Terrestrial Reference Frames (TRF) are available, the ITRF2014 from IGN, the DTRF2014 from DGFI-TUM, and JTRF2014 from JPL. All use the identical input data of space-geodetic station positions and Earth orientation parameters, but the concept of combining these data is fundamentally different. The IGN approach is based on the combination of technique solutions, while the DGFI is combining the normal equation systems. Both yield in reference epoch coordinates and velocities for a global set of stations. JPL uses a Kalman filter approach, realizing a TRF through weekly time series of geocentric coordinates. As the determination of the CRF is not independent of the TRF and vice versa, the choice of the TRF might impact on the CRF. Within this work we assess this effect. We find that the estimated Earth orientation parameter (EOP) from DTRF2014 agree best with those from ITRF2014, the EOP resulting from JTRF2014 show besides clear yearly signals also some artifacts linked to certain stations. The estimated source position time series however, agree with each other better than ±1 µas. When fixing EOP and station positions we can see the maximal effect of the TRF on the CRF. Here large systematics in position as well as proper motion arise. In case of ITRF2008 they can be linked to the missing data after 2008. By allowing the EOP and stations to participate in the adjustment, the agreement increases, however, systematics remain.

Lung transplantation after ex vivo lung perfusion in two Scandinavian centres.

OBJECTIVES: We reviewed our combined clinical outcome in patients who underwent lung transplantation after ex vivo lung perfusion (EVLP) and compared it to the contemporary control group. METHODS: At 2 Scandinavian centres, lungs from brain-dead donors, not accepted for donation but with potential for improvement, were subjected to EVLP (n = 61) and were transplanted if predefined criteria were met. Transplantation outcome was compared with that of the contemporary control group consisting of patients (n = 271) who were transplanted with conventional donor lungs. RESULTS: Fifty-four recipients from the regular waiting list underwent transplantation with lungs subjected to EVLP (1 bilateral lobar, 7 single and 46 double). In the EVLP and control groups, arterial oxygen tension/inspired oxygen fraction ratio at arrival in the intensive care unit (ICU) was 30 ± 14 kPa compared to 36 ± 14 (P = 0.005); median time to extubation was 18 h (range 2-912) compared to 7 (range 0-2280) (P = 0.002); median ICU length of stay was 4 days (range 2-65) compared to 3 days (range 1-156) (P = 0.002); Percentage of expected forced expiratory volume at 1s (FEV1.0%) at 1 year was 75 ± 29 compared to 81 ± 26 (P = 0.18); and the 1-year survival rate was 87% [confidence interval (CI) 82-92%] compared to 83% (CI 81-85), respectively. Follow-up to a maximum of 5 years did not show any significant difference in survival between groups (log rank, P = 0.63). CONCLUSIONS: Patients transplanted with lungs after EVLP showed outcomes comparable to patients who received conventional organs at medium-term follow-up. Although early outcome immediately after transplantation showed worse lung function in the EVLP group, no differences were observed at a later stage, and we consider EVLP to be a safe method for increasing the number of transplantable organs.

Comparison of two strategies for ex vivo lung perfusion.

BACKGROUND: Two clinically used strategies for ex vivo lung perfusion (EVLP) were compared in a porcine model with respect to lung function, metabolism, inflammatory response, oxidative stress, and cell viability. METHODS: Porcine lungs (n = 20) were preserved, harvested, and kept cooled for 2 hours. After randomization, EVLP was performed using a cellular perfusate and open left atrium (COA group) or an acellular perfusate and a closed left atrium (ACA group). Oxygenation (partial pressure of arterial oxygen/fraction of inspired oxygen), compliance, dead space, weight, and perfusate oncotic pressure were registered before and after a 4-hour period of reconditioning. Lung tissue samples were collected before and after EVLP for quantitative polymerase chain reaction analysis of gene expression for inflammatory markers, measurement of tissue hypoxia (hypoxia inducible factor-1α) and oxidative stress (ascorbyl radical), and viability (trypan blue staining) and lung histopathology. RESULTS: In 3 of 10 lungs undergoing EVLP in the ACA group, EVLP was terminated prematurely because of severe lung edema and inability to perfuse the lungs. There were no significant differences in changes of lung oxygenation or pulmonary vascular resistance between groups. Compliance decreased and lung weights increased in both groups, but more in the ACA group (p = 0.083 and p = 0.065, respectively). There was no obvious difference in gene expression for hypoxia inducible factor-1α, inflammatory markers, free radicals, or lung injury between groups. CONCLUSIONS: Lung edema formation and decreased lung compliance occurs with both EVLP techniques but were more pronounced in the ACA group. Otherwise, there were no differences in lung function, inflammatory response, ischemia/reperfusion injury, or histopathologic changes between the EVLP techniques.

The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate.

We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.

Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis.

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

Mimicking respiratory phosphorylation using purified enzymes.

The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Hemofiltration in ex vivo lung perfusion-a study in experimentally induced pulmonary edema.

OBJECTIVES: Ex vivo lung perfusion (EVLP) can potentially reduce pulmonary edema. In a pig model with induced pulmonary edema, we evaluated the effect of hemofiltration (HF) during EVLP on lung function, perfusate oncotic pressure, and lung weight. METHODS: In anesthetized pigs (n = 14), pulmonary edema was induced by a balloon in the left atrium, combined with crystalloid infusion (20 mL/kg), for 2 hours. The lungs were harvested, stored cold for 2 hours, and randomized to EVLP, with or without a hemofilter (HF and noHF groups, respectively, n = 7 for each). EVLP was performed with cellular perfusate at a hematocrit of 10% to 15%. Oncotic pressure, lung performance, and weight were measured before and after 180 minutes of EVLP reconditioning with or without HF. RESULTS: After in vivo induction of edema, arterial oxygen tension (Pao2)/inspired oxygen fraction (Fio2), and compliance decreased by 63% and 16%, respectively. Pao2/Fio2 was considerably improved at first evaluation ex vivo in both groups. HF increased oncotic pressure by 43% and decreased lung weight by 15%. The effects were negligible in the noHF group. Compliance decreased in both groups during reconditioning, although less so in the HF group (P < .05). Pao2/Fio2, shunt fraction, and oxygen saturation remained unchanged in both groups. Pulmonary flow index decreased in both groups, and was partially reversed by nitroglycerin. Dorsal atelectatic consolidations were seen in both groups. CONCLUSIONS: In this lung-edema model, EVLP reconditioning with hyperoncotic solution did not affect the degree of lung edema. HF during EVLP increased perfusate oncotic pressure, decreased lung weight with beneficial effects on compliance, but did not improve lung oxygenation capacity.

Fluorescent discrimination between traces of chemical warfare agents and their mimics.

An array of fluorogenic probes is able to discriminate between nerve agents, sarin, soman, tabun, VX and their mimics, in water or organic solvent, by qualitative fluorescence patterns and quantitative multivariate analysis, thus making the system suitable for the in-the-field detection of traces of chemical warfare agents as well as to differentiate between the real nerve agents and other related compounds.

New VLBI2010 scheduling strategies and implications on the terrestrial reference frames.

In connection with the work for the next generation VLBI2010 Global Observing System (VGOS) of the International VLBI Service for Geodesy and Astrometry, a new scheduling package (Vie_Sched) has been developed at the Vienna University of Technology as a part of the Vienna VLBI Software. In addition to the classical station-based approach it is equipped with a new scheduling strategy based on the radio sources to be observed. We introduce different configurations of source-based scheduling options and investigate the implications on present and future VLBI2010 geodetic schedules. By comparison to existing VLBI schedules of the continuous campaign CONT11, we find that the source-based approach with two sources has a performance similar to the station-based approach in terms of number of observations, sky coverage, and geodetic parameters. For an artificial 16 station VLBI2010 network, the source-based approach with four sources provides an improved distribution of source observations on the celestial sphere. Monte Carlo simulations yield slightly better repeatabilities of station coordinates with the source-based approach with two sources or four sources than the classical strategy. The new VLBI scheduling software with its alternative scheduling strategy offers a promising option with respect to applications of the VGOS.

Automatic measurements of diameter, distension and intima media thickness of the aorta in premature rabbit pups using B-mode images.

To improve cardiovascular research, there is a growing need for arterial characterization in small animals. We developed a method, ARTIC (arterial characterization) for measuring lumen diameter, distension and intima media thickness (IMT). In this study ARTIC was used to automatically characterize the aorta of premature rabbit pups. Automatic measurements were compared with manual measurements, both performed by three observers. Diameter was 769 ± 140 µm (manual) and 766 ± 142 µm (automatic), distension was 35 ± 15 µm (manual) and 40 ± 12 µm (automatic) and IMT was 84 ± 11 µm (manual) and 88 ± 8 µm (automatic) (mean ± standard deviation). The variation in the measured diameter, distension and IMT ranged from 1.1% to 26.0% (manual) and from 1.0% to 9.0% (automatic). The intra-class correlation coefficient ranged from 33.0% to 99.3% (manual) and from 76.9% to 99.6% (automatic). The evaluation revealed that it is feasible to use ARTIC on B-mode images of arteries with small dimensions, which makes it a useful tool for arterial characterization in small animals.

Parallel haemodialysis and surgery saves a life after massive overdose of potassium pills.

Severe poisoning with potassium pills is rare but patients may present with serious cardiovascular symptoms requiring immediate and effective treatment. A 30-year-old healthy woman presented to the emergency department after ingestion of 300 slow-release pills of potassium-chloride with serum potassium of 9.5 mmol/l, and poor cardiovascular function. Gastric lavage was performed with poor outcome. Despite intensive medical treatment serum potassium remained very high around 9 mmol/l. Haemodialysis was initiated but despite ongoing dialysis, potassium increased to 10.3 mmol/l. Hence, a parallel dialysis was started and after 4 h of parallel dialysis, serum potassium decreased to 6.4 mmol/l. An x-ray revealed large amounts of pills remaining in the stomach and the surgeon was able to remove about 200 pills through an acute laparotomy. The patient recovered slowly thereafter, but later developed a gastric stricture and pulmonary embolism.

Longitudinal displacement and intramural shear strain of the porcine carotid artery undergo profound changes in response to catecholamines.

The effects of catecholamines on longitudinal displacements and intramural shear strain of the arterial wall are unexplored. Therefore, the common carotid artery of five anaesthetized pigs was investigated using an in-house developed noninvasive ultrasonic technique. The study protocol included intravenous infusion of low-dose epinephrine (ß-adrenoceptor activation), as well as intravenous boluses of norepinephrine (α-adrenoceptor activation). Further, the effects of ß-blockade (metoprolol) were studied. There were significant positive correlations between pulse pressure and longitudinal displacement of the intima-media complex (r = 0.72; P < 0.001), as well as between pulse pressure and intramural shear strain (r = 0.48; P < 0.001). Following administration of norepinephrine, the longitudinal displacement of the intima-media complex and intramural shear strain profoundly increased (median 190%, range 102-296%, and median 141%, range 101-182%, respectively, compared with baseline), also when given during ß-blockade (median 228%, range 133-266%, and median 158%, range 152-235%, respectively). During infusion of low-dose epinephrine, the longitudinal displacement of the intima-media complex and intramural shear strain decreased (median 88%, range 69-122%, and median 69%, range 47-117%, respectively, compared with baseline). In conclusion, the present study shows, for the first time, that the longitudinal displacement and intramural shear strain of the porcine carotid artery undergo profound changes in response to catecholamines. Increase in longitudinal displacements seems to be strongly related to α-adrenoceptor activation. Thus metoprolol is insufficient to counteract a profound increase in longitudinal displacement and intramural shear strain following a surge of norepinephrine.

Particle-specific sorption/desorption properties determine test compound fate and bioavailability in toxicity tests with Chironomus riparius--high-resolution studies with lindane.

We studied the sorption (batch equilibrium experiments) and desorption (consecutively harsher supercritical fluid extractions) of lindane to different types of sediment and food particles, as well as larval uptake in standardized peat-based artificial sediment toxicity tests with the midge Chironomus riparius. Lindane sorption to organic particles was fast and efficient, reaching 98+/-0.1 and 97+/-0.1% of added compound in 48 h for peat and Tetraphyll(R), respectively, and 77+/-0.2% in whole sediment. Sorption to inorganic particles, that is, sand and kaolin clay, was much lower, 9.6+/-1.3% and 8.3+/-0.8%, respectively. Supercritical fluid extractions showed that most of the lindane sorbed to organic particles and sediment was loosely bound, as only 9 to 14% remained associated with particles after weak and intermediate extractions strengths. Larval uptake of dissolved lindane was 4.9+/-0.71 and 10.8+/-1.2 microg/g wet weight in 22 and 68 microg/L treatments, respectively, and four to five times higher than that of particle-associated lindane, ranging 1.0+/-0.15 to 2.7+/-0.21 microg/g in the above treatments. Surprisingly, larval uptake of lindane was similar from refractory peat and the more labile Tetraphyll particles. Despite an efficient larval uptake of dissolved lindane, sorption/desorption of lindane to/from Tetraphyll particles will facilitate digestive uptake in toxicity tests, particularly in spiked-water scenarios where food particles may act as vectors. Our results show that the exposure scenario is an important determinant for the behavior and bioavailability of test compounds in standardized toxicity tests.