Does hypoxia-inducible factor 1α play a role in regulating cutaneous oxygen flux in larval zebrafish (Danio rerio)?

Previous studies have demonstrated that hypoxia tolerance is improved in zebrafish (Danio rerio) larvae after prior exposure to lowered ambient O2 levels. Such improved hypoxia performance was attributed in part, to increased levels of hypoxia-inducible factor 1α (Hif-1α) exerting downstream effects on various physiological processes including promotion of trunk skin angiogenesis. Since O2 uptake ([Formula: see text]) in larvae is facilitated largely by O2 diffusion across the skin, enhanced cutaneous vascularization is expected to enhance [Formula: see text] during hypoxia and thus contribute to improved hypoxia tolerance. In this study, we used the scanning micro-optrode technique together with quantification of cutaneous vascularity in wild types (WT) and Hif-1α knockouts (hif1aa-/-ab-/-) to test the hypothesis that improved hypoxia tolerance after hypoxia acclimation in larvae at 4 or 7 days post-fertilization (dpf) was associated with Hif-1α-dependent increases in skin vascularity and regional cutaneous O2 fluxes (JO2). Hypoxia tolerance, as determined by measurements of critical PO2 (Pcrit), was unaltered by hypoxia pre-exposure in larvae at 4 dpf and there were no significant differences in Pcrit between WT and hif1aa-/-ab-/- larvae at this developmental stage. However, at 7 dpf there was a significant effect of genotype with WT larvae showing a lower Pcrit than hif1aa-/-ab-/- larvae, an effect that was being driven by a reduced Pcrit in the WT larvae after hypoxia pre-exposure (19.2 ± 1.9 mmHg) compared to hif1aa-/-ab-/- fish (35.5 ± 3.5 mmHg). Regardless of genotype, pre-exposure status or developmental age, JO2 decreased along the body in the anterior-to-posterior direction. Neither hypoxia pre-exposure nor genotype affected JO2 at any region along the body. The lack of any effect of hypoxia pre-exposure or genotype on JO2 was consistent with the lack of any effect on skin vascularity as measured in Tg(fli1:EGFP)yl transgenic larvae. Thus, the decreased hypoxia performance (increased Pcrit) at 7 dpf in the hif1aa-/-ab-/- larvae did not appear to be reliant on changes in trunk vascularity or cutaneous O2 diffusion.

Multicohort Retrospective Validation of a Predictive Biomarker for Topoisomerase I Inhibitors.

PURPOSE: The camptothecin (CPT) analogs topotecan and irinotecan specifically target topoisomerase I (topoI) and are used to treat colorectal, gastric, and pancreatic cancer. Response rate for this class of drug varies from 10% to 30%, and there is no predictive biomarker for patient stratification by response. On the basis of our understanding of CPT drug resistance mechanisms, we developed an immunohistochemistry-based predictive test, P-topoI-Dx, to stratify the patient population into those who did and did not experience a response. PATIENTS AND METHODS: The retrospective validation studies included a training set (n = 79) and a validation cohort (n = 27) of gastric cancer (GC) patients, and 8 cohorts of colorectal cancer (CRC) patient tissue (n = 176). Progression-free survival for 6 months was considered a positive response to CPT-based therapy. Formalin-fixed, paraffin-embedded slides were immunohistochemically stained with anti-phospho-specific topoI-Serine10 (topoI-pS10), quantitated, and analyzed statistically. RESULTS: We determined a threshold of 35% positive staining to offer optimal test characteristics in GC. The GC (n = 79) training set demonstrated 76.6% (95% confidence interval, 64-86) sensitivity; 68.8% (41-88) specificity; positive predictive value (PPV) 92.5% (81-98); and negative predictive value (NPV) 42.3% (24-62). The GC validation set (n = 27) demonstrated 82.4% (56-95) sensitivity and 70.0% (35-92) specificity. Estimated PPV and NPV were 82.4% (56-95) and 70.0% (35-92) respectively. In the CRC validation set (n = 176), the 40% threshold demonstrated 87.5% (78-94) sensitivity; 70.0% (59-79) specificity; PPV 70.7% (61-79); and NPV 87.0 % (77-93). CONCLUSION: The analysis of retrospective data from patients (n = 282) provides clinical validity to our P-topoI-Dx immunohistochemical test to identify patients with disease that is most likely to respond to topoI inhibitors.

Respirometry and cutaneous oxygen flux measurements reveal a negligible aerobic cost of ion regulation in larval zebrafish (Danio rerio).

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish (Danio rerio). We hypothesized that changes in rates of Na+ uptake evoked by acidic or low Na+ rearing conditions would result in corresponding changes in whole-body oxygen consumption (MO2 ) and/or cutaneous oxygen flux (JO2 ), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na+ uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower MO2  and no difference in cutaneous JO2 Despite a higher Na+ uptake capacity in larvae reared under low Na+ conditions, there were no differences in MO2  and JO2  at 4 dpf. Furthermore, although Na+ uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a, MO2 and JO2  were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous JO2 Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na+, are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost.

Developing a Phosphospecific IHC Assay as a Predictive Biomarker for Topoisomerase I Inhibitors.

Phosphorylation is the most extensively studied posttranslational modification of proteins. There are approximately 500 kinases known in the human genome. The kinase-activated pathways regulate almost every aspect of cell function and a deregulated kinase cascade leads to impaired cellular function. Impaired regulation of several kinase cascades, including the epidermal growth factor receptor (EGFR) pathway, leading to tumor pathogenesis, is well documented. Thus, a phosphospecific test with prognostic or predictive value was expected in oncology. However, no phosphospecific IHC test is used in oncology clinics. Human topoisomerase I (topoI) inhibitors, camptothecin and its analogues (CPT), are used extensively to treat various solid tumors. Depending on tumor type, the response rate is only 13-32%. We have demonstrated that the deregulated kinase cascade is at the core of CPT resistance. DNA-PKcs, a kinase central to the DNA-double-strand break (DSB) response pathway, phosphorylates topoI at serine 10 (topoI-pS10), and cells with higher basal levels of topoI-pS10 degrade topoI rapidly and are resistant to this class of drug. The higher basal level of topoI phosphorylation is due to continual activation of DNA-PKcs, and one potential mechanism of this pathway activation is failure of upstream effector phosphatases such as phosphatase and tensin homolog (PTEN). Based on this understanding, we have developed an IHC-based test (P-topoIDx) that can stratify the responder and non-responder patient population.

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

Hygiene and sanitation practices amongst residents of three long-term refugee camps in Thailand, Ethiopia and Kenya.

OBJECTIVE: To further the understanding of sanitation and hygiene in long-term camp populations. METHODS: Data were collected by structured observation of handwashing (126 households), a questionnaire on sanitation, hygiene and household characteristics (1089 households) and discussions with mothers. Random walk algorithms were used to select households for observation and survey. Respondents for qualitative methods were a convenience sample. RESULTS: Across all key handwash occasions [excluding events with no handwash (n=275)], soap was used for 30% of handwashes. After latrine use, both hands were washed with soap on 20% of occasions observed. Availability of soap in households differed across sites and mirrored the extent to which it was distributed free of charge. Qualitative data suggested lack of free soap as a barrier to 'safe' handwashing. Laundry was the priority for soap. In Ethiopia and Kenya, open defecation was practised by a significant minority and was more prevalent amongst households of rural origin. In Ethiopia, open defecation was significantly more prevalent amongst women. CONCLUSIONS: Despite continuing hygiene education, rates of 'safe' handwashing are sub-optimal. Soap scarcity in some households and the prioritisation of laundry are barriers to safe practice. Heterogeneity with respect to education and place of origin may need to be taken into account in the design of improved interventions.

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry.

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.

A late Triassic impact ejecta layer in southwestern Britain.

Despite the 160 or so known terrestrial impact craters of Phanerozoic age, equivalent ejecta deposits within distal sedimentary successions are rare. We report a Triassic deposit in southwestern Britain that contains spherules and shocked quartz, characteristic of an impact ejecta layer. Inter- and intragranular potassium feldspar from the deposit yields an argon-argon age of 214 +/- 2.5 million years old. This is within the age range of several known Triassic impact craters, the two closest of which, both in age and location, are Manicouagan in northeastern Canada and Rochechouart in central France. The ejecta deposit provides an important sedimentary record of an extraterrestrial impact in the Mesozoic that will help to decipher the number and effect of impact events, the source and dynamics of the event that left this distinctive sedimentary marker, and the relation of this ejecta layer to the timing of extinctions in the fossil record.