Innovative snow harvesting technology increases vegetation establishment success in native sagebrush ecosystem restoration

Aims

In cold-arid systems, moisture availability is often a limiting factor to vegetation establishment and growth. Consequentially, manipulating soil moisture dynamics may help to improve restoration practice. Application of snow fencing, for microclimate manipulation in cold-arid regions, improves native species establishment and restoration success following disturbance by improving soil moisture retention.

Methods

Research was conducted in the Jonah Natural Gas Field, an area subjected to intensive gas development, in southwest Wyoming, USA, to assess roles of snow fencing in altering snowpack characteristics, and soil moisture, for improving re-establishment of native vegetation.

Results

Application of a customized snow fence design (the Hollow Frame Fence System, HFFS) significantly increased vegetation establishment of the framework taxon, Artemisia tridentata Nutt. spp. wyomingensis Beetle & Young, and other native sagebrush-steppe species. The HFFS exhibited significantly fewer invasive species than control blocks; establishment of A. tridentata was significantly higher within the HFFS, as compared to both control blocks, and commercial fence types. Significant increases in spring soil moisture are likely drivers of increased restoration success with use of HFFS.

Conclusions

The Hollow Frame Fence System provides increased water harvesting capacity necessary to improve habitat restoration in cold-arid environments subjected to disturbance.     

Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules

The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5′ end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3′ noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.     

What a Difference a Dalton Makes: Bacterial Virulence Factors Modulate Eukaryotic Host Cell Signaling Systems via Deamidation

SUMMARY

Pathogenic bacteria commonly deploy enzymes to promote virulence. These enzymes can modulate the functions of host cell targets. While the actions of some enzymes can be very obvious (e.g., digesting plant cell walls), others have more subtle activities. Depending on the lifestyle of the bacteria, these subtle modifications can be crucially important for pathogenesis. In particular, if bacteria rely on a living host, subtle mechanisms to alter host cellular function are likely to dominate. Several bacterial virulence factors have evolved to use enzymatic deamidation as a subtle posttranslational mechanism to modify the functions of host protein targets. Deamidation is the irreversible conversion of the amino acids glutamine and asparagine to glutamic acid and aspartic acid, respectively. Interestingly, all currently characterized bacterial deamidases affect the function of the target protein by modifying a single glutamine residue in the sequence. Deamidation of target host proteins can disrupt host signaling and downstream processes by either activating or inactivating the target. Despite the subtlety of this modification, it has been shown to cause dramatic, context-dependent effects on host cells. Several crystal structures of bacterial deamidases have been solved. All are members of the papain-like superfamily and display a cysteine-based catalytic triad. However, these proteins form distinct structural subfamilies and feature combinations of modular domains of various functions. Based on the diverse pathogens that use deamidation as a mechanism to promote virulence and the recent identification of multiple deamidases, it is clear that this enzymatic activity is emerging as an important and widespread feature in bacterial pathogenesis.     

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30 °C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30 °C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30 °C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.     

Crab regulation of cross-ecosystem resource transfer by marine foraging fire ants

Permeability of boundaries in biological systems is regulated by biotic and/or abiotic factors. Despite this knowledge, the role of biotic factors in regulating resource transfer across ecosystem boundaries has received little study. Additionally, little is known about how cross-ecosystem resource transfer affects source populations. We used experiments, observations and stable isotopes, to evaluate: (1) the proportion of intertidal-foraging black fire ant (Solenopsis richteri) diet derived from marine sources, (2) how black fire ant cross-ecosystem resource transfer is altered by the dominant bioengineer in the intertidal, a burrowing crab (Neohelice granulata), (3) the top-down impact of these terrestrial ants on a marine resource, and (4) the effect of marine resources on recipient black fire ants. We found that more than 85% of the black fire ant diet is derived from marine sources, the number of intertidal foraging ants doubles in the absence of crab burrows, and that ants cause a 50% reduction in intertidal polychaetes. Also, ant mound density is three times greater adjacent to marine systems. This study reveals that cross-ecosystem foraging terrestrial ants can clearly have strong impacts on marine resources. Furthermore, ecosystem engineers that modify and occupy habitat in these ecosystem boundaries can strongly regulate the degree of cross-ecosystem resource transfer and resultant top down impacts.     

Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 Å, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU''s preference for Ub and Ub-like substrates.Crimean-Congo hemorrhagic fever (CCHF) is characterized in humans by the sudden onset of fever, myalgia, headache, dizziness, sore eyes, photophobia, and hyperanemia as well as severe hemorrhages (28, 43, 46). The causative agent of CCHF is the CCHF virus, which is a tick-borne, negative-sense, single-stranded RNA [ssRNA(−)] virus of the genus Nairovirus, belonging to the viral family Bunyaviridae. Originally named after outbreaks in the former Soviet Union and in the Congo during the mid-20th century, the affected area of this disease has rapidly spread to large areas of sub-Saharan Africa, the Balkans, Northern Greece, European Russia, Pakistan, the Arabian Peninsula, Iran, Afghanistan, Iraq, Turkey, and recently, the Xinjiang province of China (43, 46). The CCHF viral genome, as well as those of the closely related Dugbe and Nairobi viruses, consists of three negative-sense RNA segments, small (S), medium (M), and large (L). Incubation of CCHF is 5 to 6 days, with fatalities occurring less than 7 days after signs of infection. Fatality rates for patients infected with the CCHF virus ranged from 5% to 70%, depending on phylogenetic variation of the virus, transmission route, treatment facility, and the reporting and confirmation of the case statistics (19, 32, 43, 47).The innate immune system serves as the human''s first line of defense from invading pathogens, including CCHF virus. The type I interferon (IFN) response comprises a key component of this system by upregulating more than 300 IFN-stimulated genes (ISGs) whose products detect viral molecules, promote amplification of the type I IFN response, modulate other signaling pathways, and directly provide antiviral activity (34). Regulation of the type I IFN response has been shown to rely on posttranslational modification by ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15) (14, 23). Both Ub and ISG15 are expressed in a proform and cleaved to leave a double-glycine C terminus that forms an isopeptide bond with predominantly the ɛ-NH₂ of lysine residues of a target protein through a three-step enzymatic process. In addition to forming isopeptide bonds with target proteins, Ub, which contains seven lysine residues, has been observed to form poly-Ub chains. The most studied of these moieties are K29-linked, K48-linked, and K63-linked poly-Ub. While K29-linked and K48-linked polyubiquitination of proteins leads to their degradation in the lysosome and proteasome, respectively, conjugation of K63-linked poly-Ub to proteins has an activating effect, resulting in an enhanced type I IFN response (2, 7, 18, 33, 40). Currently, more than 150 proteins have been identified as forming conjugates with ISG15, with the number of proteins forming Ub conjugates far exceeding that number (12, 48). A subset of type I IFN signaling and effector proteins that Ub and ISG15 have been shown to stabilize includes JAK1, STAT1/2, double-stranded RNA-dependent protein kinase (PKR), myxovirus-resistant protein A (MxA), and RIG-I (17). MxA has particularly shown to be important in type I IFN response to CCHF infection. RIG-I and several other proteins have also been shown to be targets for K63-linked poly-Ub (4).Recently, investigators have identified a cysteine viral ovarian tumor domain (vOTU) protease colocated with the RNA-dependent RNA polymerase in the L protein of the CCHF virus (14). Interestingly, as CCHF is an ssRNA(−) virus, no protease is required to cleave a viral polypeptide to facilitate viral replication as in positive-sense ssRNA [ssRNA(+)] viruses. Furthermore, recent reports have observed that vOTU is not required for RNA-dependent RNA polymerase activity and for vOTU protease activity linked to impairment of the type I IFN response through its deubiquitinating and deISGylating activity (6, 14). Additional studies have also tentatively identified the presence of vOTU homologues in the Arterivirus genus of the Arteriviridae family, suggesting that they too may facilitate impairment of the type I IFN response (14). Since the discovery of the first ovarian tumor domain (OTU) protease in Drosophila oogenesis and prior to the identification of vOTU, OTU superfamily members could be divided into three subclasses according to their sequence homology, otubains, A20-like OTUs, and ubiquitin thioesterase ZRANB1 (22). With the addition of the viral OTU subclass, OTU superfamily members in more than 100 eukaryotic, bacterial, and viral proteins have now been identified (6, 27). Predominantly, OTU proteases have been linked to ubiquitin (Ub) removal and/or remodeling of Ub-conjugated proteins, placing them among five protease superfamilies that facilitate signal transduction cascades and play key roles in protein stability (22). However, vOTU is unique in that it is the only OTU to have shown both deubiquitinating and deISGylating activity (14). Instead, Otubain1/2 (OTUB1/2) plays a key role in T cell response and prefers K48-linked poly-Ub or NEDD8 (neural precursor cell expressed, developmentally downregulated 8) as a substrate (12). A20 and A20-like Cezanne OTU proteases are negative regulators of the NF-κB-mediated inflammation response, selectively cleaving K63-linked poly-Ub targets. DUBA also shows preference for K63-linked poly-Ub (20). In attempts to better understand the OTU superfamily, structures of OTUB and A20-like OTU domains have been elucidated (12, 21, 30). An X-ray structure of the yeast ovarian tumor 1 (yOTU1) domain, which interacts with Cdc48 and has a preference for K48-linked poly-Ub, was achieved in complex with mono-Ub (27). However, since yOTU1 has a preference for K48-linked Ub and possesses low sequence identity to vOTU and other OTU domain proteases, only limited information on vOTU could be obtained. In addition to vOTU, several other viral proteases, such as papain-like protease (PLpro) from the severe acute respiratory syndrome (SARS) coronavirus, have also shown deubiquitinating and deISGylating activity to evade the innate immune system (6, 8, 43, 49). However, no viral proteases that are known to possess deISGylating activity have been visualized as being bound to Ub or Ub-like substrates. To address this issue and elucidate the atomic-level structure of a member from the viral OTU superfamily subclass, we have obtained the X-ray crystal structure of vOTU bound with Ub (vOTU-Ub). We also have characterized the vOTU substrate specificity for mono-Ub, ISG15, and NEDD8 and compared the results with those from human OTUB2 (hOTUB2). Additionally, we assessed vOTU''s deubiquitinating activity toward K48- and K63-linked poly-Ub.     

Interferon regulatory factor 4 (IRF-4) targets IRF-5 to regulate Epstein-Barr virus transformation

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdown-mediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation.     

Receptor-isoform-selective insulin analogues give tissue-preferential effects

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood-glucose-lowering effect upon acute intravenous dosing of mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR-isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on IR-A/IR-B expression.