Renal safety of a tenofovir-containing first line regimen: experience from an antiretroviral cohort in rural Lesotho

Introduction

Current guidelines contraindicate TDF use when creatinine clearance (CrCl) falls below 50 ml/min. We report prevalence of abnormal renal function at baseline and factors associated with abnormal renal function from a community cohort in Lesotho.

Methods

We calculated changes in CrCl from baseline for patients initiated on TDF at 6 and 12 months and the proportion of patients initiated on TDF who developed renal impairment. Screening algorithms were developed using risk factors determined by multivariate analysis.

Results

Among 933 adults for whom baseline creatinine was available, 176 (18.9%) presented with a baseline CrCl <50 ml/min. Renal function improved during follow-up. 19 patients who developed renal toxicity during follow up remained on TDF; renal function improved (CrCl≥50 ml/min) in all but 3 of these patients. Among 15 patients with a baseline CrCl <50 ml/min were started in error, none developed severe renal impairment.

Conclusion

In this setting TDF-associated renal toxicity is rare and mainly transient. Further studies to assess TDF safety at lower CrCl thresholds are warranted.     

Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models

In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand–receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.     

Infrared video thermography: a technique for assessing cold adaptation in insects

Insects can survive subzero temperatures by two main strategies: freeze tolerance and freeze avoidance. An array of techniques have been used to investigate the physiological limits of insects to low temperatures, such as differential scanning calorimetry, temperature-controlled cooling apparatus, thermocouples, and computer-controlled chart recording equipment. However, these techniques require animals to be stationary, precluding behavioral data. We used infrared video thermography to investigate cold adaptation in an alpine insect, expanding such investigations to include behavioral response as an indicator of physiological stress. This technique is noninvasive and provides a large amount of physiological information, such as supercooling points, lower lethal temperatures, and hemolymph melting points. Insect supercooling points in response to a constant cooling rate were variable; however, temperatures at the initiation of behavioral stress response were less variable. Assessments of supercooling points and lower lethal temperatures obtained in this way are more biologically meaningful because allowing unhindered movement of insects more closely resembles natural environments.     

Experimentally elevated testosterone increases status signalling in male Greylag geese (<Emphasis Type="Italic">Anser anser</Emphasis>)

Testosterone modulates male vertebrates sexual and social behaviour. We experimentally investigated the testosterone-sensitive behaviours in male greylag geese (Anser anser) by implanting silastic tubes containing crystalline testosterone during the mating season (February; 5 implanted and 5 control males) and in the early winter (November; 7 and 7). Focal animals were part of a semi-tame, unrestrained flock with fully intact social relationships. Excreted testosterone and corticosterone immunoreactive metabolites (TM, BM) were determined by enzyme immunoassay. Individual faecal samples and behavioural protocols were collected daily over a period of 5 weeks, including 1 control week before implantation. In February, no significant behavioural effects of the supplemental testosterone were observed, which may be due to the naturally occurring high systemic androgen levels in spring. In November, however, implanted males had higher TM excretion rates and performed status signalling behaviour (beak up) more frequently than control males. No differences between implanted and control males were found with respect to BM, agonistic interactions or vigilance behaviour. Furthermore, during the second week after implantation, TM positively correlated with the frequency of beak up of implanted males, whilst their female partners were attacked with lower latency by other members of the flock than the females of control males. Hence, status signalling in greylag ganders seems to be testosterone-sensitive year-long and inappropriate status signalling of males may draw attacks towards their females.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

Contribution of the cytoskeleton and the phospholipase C signaling pathway to fluid stream-induced membrane currents

A fluid stream induced by a concentration clamp system evokes in Xenopus oocytes a deformation of the membrane which results in transient chloride currents of high amplitude (stream-evoked inward current, I(i,st)) during calcium-activated chloride current oscillations. The involvement of cytoskeleton elements and of components of the phospholipase C-dependent signaling pathway on the generation of the I(i,st) were investigated. Incubation of the oocytes with cytoskeleton-disrupting agents exerted no effects on generation of the I(i,st), suggesting that the mechanotransduction is not mediated by these structures. The fluid stream induced an elevation of the submembraneous calcium concentration, as measured by an increase of Fluo-4-mediated fluorescence after the stimulus. Lowering the intracellular calcium concentration by injection of calcium chelators or depleting inositol 1,4,5-triphosphate (InsP(3))-sensitive calcium stores by blockers of the calcium pumps suppressed the generation of the I(i,st) in most cases. Furthermore, the phospholipase C inhibitor U73122 reversibly blocked the I(i,st). The results suggest that the fluid stream leads to a membrane stretch which modulates directly or indirectly the activity of a membrane-bound phospholipase C. The phospholipase C transiently elevates the InsP(3) concentration, in turn releasing calcium from InsP(3)-sensitive internal calcium stores, thus evoking an enhanced calcium-sensitive chloride current.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.