ADAM10 Is the Major Sheddase Responsible for the Release of Membrane-associated Meprin A

Meprin A, composed of α and β subunits, is a membrane-bound metalloproteinase in renal proximal tubules. Meprin A plays an important role in tubular epithelial cell injury during acute kidney injury (AKI). The present study demonstrated that during ischemia-reperfusion-induced AKI, meprin A was shed from proximal tubule membranes, as evident from its redistribution toward the basolateral side, proteolytic processing in the membranes, and excretion in the urine. To identify the proteolytic enzyme responsible for shedding of meprin A, we generated stable HEK cell lines expressing meprin β alone and both meprin α and meprin β for the expression of meprin A. Phorbol 12-myristate 13-acetate and ionomycin stimulated ectodomain shedding of meprin β and meprin A. Among the inhibitors of various proteases, the broad spectrum inhibitor of the ADAM family of proteases, tumor necrosis factor-α protease inhibitor (TAPI-1), was most effective in preventing constitutive, phorbol 12-myristate 13-acetate-, and ionomycin-stimulated shedding of meprin β and meprin A in the medium of both transfectants. The use of differential inhibitors for ADAM10 and ADAM17 indicated that ADAM10 inhibition is sufficient to block shedding. In agreement with these results, small interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited meprin β and meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhanced shedding of meprin β from both transfectants. Our studies demonstrate that ADAM10 is the major ADAM metalloproteinase responsible for the constitutive and stimulated shedding of meprin β and meprin A. These studies further suggest that inhibiting ADAM 10 activity could be of therapeutic benefit in AKI.     

Effects of urban stream burial on nitrogen uptake and ecosystem metabolism: implications for watershed nitrogen and carbon fluxes

Urbanization has resulted in the extensive burial and channelization of headwater streams, yet little is known about the impacts of stream burial on ecosystem functions critical for reducing downstream nitrogen (N) and carbon (C) exports. In order to characterize the biogeochemical effects of stream burial on N and C, we measured NO₃ ^? uptake (using ¹⁵N-NO₃ ^? isotope tracer releases) and gross primary productivity (GPP) and ecosystem respiration (ER) (using whole stream metabolism measurements). Experiments were carried out during four seasons, in three paired buried and open stream reaches, within the Baltimore Ecosystem Study Long-term Ecological Research site. Stream burial increased NO₃ ^? uptake lengths by a factor of 7.5 (p < 0.01) and decreased NO₃ ^? uptake velocity and areal NO₃ ^? uptake rate by factors of 8.2 (p < 0.05) and 9.6 (p < 0.001), respectively. Stream burial decreased GPP by a factor of 11.0 (p < 0.01) and decreased ER by a factor of 5.0 (p < 0.05). From fluorescence Excitation Emissions Matrices analysis, buried streams were found to have significantly altered C quality, showing less labile dissolved organic matter. Furthermore, buried streams had significantly lower transient storage (TS) and water temperatures. Differences in NO₃ ^? uptake, GPP, and ER in buried streams, were primarily explained by decreased TS, light availability, and C quality, respectively. At the watershed scale, we estimate that stream burial decreases NO₃ ^? uptake by 39 % and C production by 194 %. Overall, our results suggest that stream burial significantly impacts NO₃ ^? uptake, stream metabolism, and the quality of organic C exported from watersheds. Given the large impacts of stream burial on stream ecosystem processes, daylighting or de-channelization of streams, through hydrologic floodplain reconnection, may have the potential to alter ecosystem functions in urban watersheds, when used appropriately.     

Effects of stormwater management and stream restoration on watershed nitrogen retention

Restoring urban infrastructure and managing the nitrogen cycle represent emerging challenges for urban water quality. We investigated whether stormwater control measures (SCMs), a form of green infrastructure, integrated into restored and degraded urban stream networks can influence watershed nitrogen loads. We hypothesized that hydrologically connected floodplains and SCMs are “hot spots” for nitrogen removal through denitrification because they have ample organic carbon, low dissolved oxygen levels, and extended hydrologic residence times. We tested this hypothesis by comparing nitrogen retention metrics in two urban stream networks (one restored and one urban degraded) that each contain SCMs, and a forested reference watershed at the Baltimore Long-Term Ecological Research site. We used an urban watershed continuum approach which included sampling over both space and time with a combination of: (1) longitudinal reach-scale mass balances of nitrogen and carbon conducted over 2 years during baseflow and storms (n = 24 sampling dates × 15 stream reaches = 360) and (2) ¹⁵N push–pull tracer experiments to measure in situ denitrification in SCMs and floodplain features (n = 72). The SCMs consisted of inline wetlands installed below a storm drain outfall at one urban site (restored Spring Branch) and a wetland/wet pond configured in an oxbow design to receive water during high flow events at another highly urbanized site (Gwynns Run). The SCMs significantly decreased total dissolved nitrogen (TDN) concentrations at both sites and significantly increased dissolved organic carbon concentrations at one site. At Spring Branch, TDN retention estimated by mass balance (g/day) was ~150 times higher within the stream network than the SCMs. There were no significant differences between mean in situ denitrification rates between SCMs and hydrologically connected floodplains. Longitudinal N budgets along the stream network showed that hydrologically connected floodplains were important sites for watershed nitrogen retention due to groundwater–surface water interactions. Overall, our results indicate that hydrologic variability can influence nitrogen source/sink dynamics along engineered stream networks. Our analysis also suggests that some major predictors for watershed N retention were: (1) streamwater and groundwater flux through stream restoration or stormwater management controls, (2) hydrologic residence times, and (3) surface area of hydrologically connected features.     

LIN28A Expression Reduces Sickling of Cultured Human Erythrocytes

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.     

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section¹. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).     

Human apolipoprotein E4 targeted replacement in mice reveals increased susceptibility to sleep disruption and intermittent hypoxia

Intermittent hypoxia (IH) and sleep fragmentation (SF) are major manifestations of sleep apnea, a frequent condition in aging humans. Sleep perturbations are frequent in Alzheimer's disease (AD) and may underlie the progression of disease. We hypothesized that acute short-term IH, SF, and their combination (IH+SF) may reveal unique susceptibility in sleep integrity in a murine model of AD. The effects of acute IH, SF, and IH+SF on sleep architecture, delta power, sleep latency, and core body temperature were assessed in adult male human ApoE4-targeted replacement mice (hApoE4) and wild-type (WT) controls. Slow wave sleep (SWS) was significantly reduced, and rapid eye movement (REM) sleep was almost abolished during acute exposure to IH alone and IH+SF for 6 h in hApoE4, with milder effects in WT controls. Decreased delta power during SWS did not show postexposure rebound in hApoE4 unlike WT controls. IH and IH+SF induced hypothermia, which was more prominent in hApoE4 than WT controls. Mice subjected to SF also showed sleep deficits but without hypothermia. hApoE4 mice, unlike WT controls, exhibited increased sleep propensity, especially following IH and IH+SF, suggesting limited ability for sleep recovery in hApoE4 mice. These findings substantiate the potential impact of IH and SF in modulating sleep architecture and sleep homeostasis including maintenance of body temperature. Furthermore, the increased susceptibility and limited recovery ability of hApoE4 mice to sleep apnea suggests that early recognition and treatment of the latter in AD patients may restrict the progression and clinical manifestations of this frequent neurodegenerative disorder.     

Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates

Background

Although it has proven to be an important foundation for investigations of carnivoran ecology, biology and evolution, the complete species-level supertree for Carnivora of Bininda-Emonds et al. is showing its age. Additional, largely molecular sequence data are now available for many species and the advancement of computer technology means that many of the limitations of the original analysis can now be avoided. We therefore sought to provide an updated estimate of the phylogenetic relationships within all extant Carnivora, again using supertree analysis to be able to analyze as much of the global phylogenetic database for the group as possible.

Results

In total, 188 source trees were combined, representing 114 trees from the literature together with 74 newly constructed gene trees derived from nearly 45,000 bp of sequence data from GenBank. The greater availability of sequence data means that the new supertree is almost completely resolved and also better reflects current phylogenetic opinion (for example, supporting a monophyletic Mephitidae, Eupleridae and Prionodontidae; placing Nandinia binotata as sister to the remaining Feliformia). Following an initial rapid radiation, diversification rate analyses indicate a downturn in the net speciation rate within the past three million years as well as a possible increase some 18.0 million years ago; numerous diversification rate shifts within the order were also identified.

Conclusions

Together, the two carnivore supertrees remain the only complete phylogenetic estimates for all extant species and the new supertree, like the old one, will form a key tool in helping us to further understand the biology of this charismatic group of carnivores.     

Plant regeneration in <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> from cell suspension cultures

A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without agar. Single cells were isolated after 3 d and the optimum cell density was 1–3 × 10⁴ cells per cm³ of the liquid MS medium. Plating efficiency was 29.6 % and callus formed within 4 weeks was subcultured and transferred to solid MS medium supplemented with 0.6 mg dm⁻³ benzyladenine (BA) along with 0.05 mg dm⁻³ α-naphthalene-1-acetic acid (NAA) for the induction of adventitious bud primordia. The shoots developed were isolated and re-cultured on MS medium containing 0.6 mg dm⁻³ BA. These microshoots after dipping in 1–2 cm³ of 10 mg dm⁻³ indole-3-butyric acid (IBA) for 24 h in dark were cultured on half strength solid MS medium supplemented with 0.05 % charcoal and showed 80–82 % rooting within 4 weeks.     

Long-term hypoxia modulates expression of key genes regulating adipose function in the late-gestation ovine fetus

A major function of abdominal adipose in the newborn is nonshivering thermogenesis. Uncoupling protein (UCP) UCP1 and UCP2 play major roles in thermogenesis. The present study tested the hypothesis that long-term hypoxia (LTH) modulates expression of UCP1 and UCP2, and key genes regulating expression of these genes in the late-gestation ovine fetus. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dG); perirenal adipose tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dG. Quantitative real-time PCR was used to analyze mRNA for UCP1, UCP2, 11beta hydroxysteroid dehydrogenase type 1 (HSD11B1) and 2 (HSD11B2), glucocorticoid receptor (GR), beta3 adrenergic receptor (beta3AR), deiodinase type 1 (DIO1) and DIO2, peroxisome proliferator activated receptor (PPAR) alpha and gamma and PPARgamma coactivator 1 (PGC1alpha). Concentrations of mRNA for UCP1, HSD11B1, PPARgamma, PGC1, DIO1, and DIO2 were significantly higher in perirenal adipose of LTH compared with control fetuses, while mRNA for HSD11B2, GR, or PPARalpha in perirenal adipose did not differ between control and LTH fetuses. The increased expression of UCP1 is likely an adaptive response to LTH, assuring adequate thermogenesis in the event of birth under oxygen-limiting conditions. Because both glucocorticoids and thyroid hormone regulate UCP1 expression, the increase in HSD11B1, DIO1, and DIO2 implicate increased adipose capacity for local synthesis of these hormones. PPARgamma and its coactivator may provide an underlying mechanism via which LTH alters development of the fetal adipocyte. These findings have important implications regarding fetal/neonatal adipose tissue function in response to LTH.