Peptide-based activation of alpha5 integrin for promoting osteogenesis

Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.     

A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A

Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z' factor of ≥ 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.     

The Dac-tag, an affinity tag based on penicillin-binding protein 5

Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.     

Changes in Serum Zinc Levels Associated with Giardiasis and Dietary Zinc Intake in Mice

The association of giardiasis with the malabsorption of zinc remains controversial. This study investigated changes in serum zinc levels in Giardia-infected mice subjected to different dietary zinc regimens. Thirty-five mice (strain C₃H/H_eJ) were randomly categorized into two groups. The first group was inoculated with 5 × 10⁶ Giardia trophozoites (n = 18), and the second group remained Giardia free (n = 17). Each group (Giardia infected and Giardia free) was randomly classified into three subgroups and given low (9 mg Zn/kg), normal (33 mg Zn/kg), and high levels (288 mg Zn/kg) of dietary zinc over a 2-week period for acclimation. Fourteen days post-Giardia infection, all of the mice were euthanized and blood samples were collected. The number of trophozoites was quantified (hematocytometer), and serum zinc levels were determined via atomic absorption spectrophotometry. Significant increases in the median weights were only found in the Giardia-free mice (p < 0.05). A higher final median weight was found in the Giardia-free group when compared with that of the Giardia-infected group given low dietary zinc (p = 0.013). In the Giardia-infected group with low dietary zinc, the geometric mean of trophozoites was 3,498 ± 101 (SE) per milliliter. The Giardia-infected group had lower serum zinc levels than did the Giardia-free group with the high dietary zinc regimens (p < 0.05). Our results are consistent with studies among human populations, but further studies are required to elucidate the actual mechanism governing the zinc–giardiasis interaction.     

Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill

The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5°C, CO(2) evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl(2) addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl(2) . Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5°C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.     

Population parameters and conservation implications for one of the world's rarest marine fishes,the red handfish (Thymichthys politus)

Population estimates are required for effective conservation of many rare marine species, but can be difficult to obtain. The critically endangered red handfish (Thymichthys politus) is a coastal anglerfish known only from two fragmented populations in southeast Tasmania, Australia. It is at a high risk of extinction due to low numbers, loss of habitat, and the impacts of climate change. To aid conservation efforts, we provide the first empirical population size estimates of red handfish and investigate other important aspects of the species' life history, such as growth, habitat association, and movement. We surveyed both red handfish local populations via underwater visual census on scuba over 3 years and used photographic mark-recapture techniques to estimate biological parameters. In 2020, the local adult population size was estimated to be 94 (95% confidence interval [CI] 40–231) adults at one site, and 7 (95% CI 5–10) at the other site, suggesting an estimated global population of 101 adults. Movement of individuals was extremely limited at 48.5 m (± 77.7 S.D. ) per year. We also found evidence of declining fish density, a declining proportion of juveniles, and increasing average fish size during the study. These results provide a serious warning that red handfish are likely sliding toward extinction, and highlight the urgent need to expand efforts for ex situ captive breeding to bolster numbers in the wild and maintain captive insurance populations, and to protect vital habitat to safeguard the species' ongoing survival in the wild.