Population genomic evidence for plant glacial survival in Scandinavia

Quaternary glaciations have played a major role in shaping the genetic diversity and distribution of plant species. Strong palaeoecological and genetic evidence supports a postglacial recolonization of most plant species to northern Europe from southern, eastern and even western glacial refugia. Although highly controversial, the existence of small in situ glacial refugia in northern Europe has recently gained molecular support. We used genomic analyses to examine the phylogeography of a species that is critical in this debate. Carex scirpoidea Michx subsp. scirpoidea is a dioecious, amphi‐Atlantic arctic–alpine sedge that is widely distributed in North America, but absent from most of Eurasia, apart from three extremely disjunct populations in Norway, all well within the limits of the Weichselian ice sheet. Range‐wide population sampling and variation at 5,307 single nucleotide polymorphisms show that the three Norwegian populations comprise unique evolutionary lineages divergent from Greenland with high between‐population divergence. The Norwegian populations have low within‐population genetic diversity consistent with having experienced genetic bottlenecks in glacial refugia, and host private alleles that probably accumulated in long‐term isolated populations. Demographic analyses support a single, pre‐Weichselian colonization into Norway from East Greenland, and subsequent divergence of the three populations in separate refugia. Other refugial areas are identified in North‐east Greenland, Minnesota/Michigan, Colorado and Alaska. Admixed populations in British Columbia and West Greenland indicate postglacial contact. Taken together, evidence from this study strongly indicates in situ glacial survival in Scandinavia.     

Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase. Expression of the latent zymogen in Pichia pastoris,autolytic activation,and the peptide sequence of the cleavage forms

An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112). The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.     

The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.     

Thermodynamic stability of bovine α-crystallin in its interactions with other bovine crystallins

Light scattering measurements were performed on dilute solutions of α-crystallin mixed with different combinations of β_H, β_L and γ-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of α-crystallin is progressively enhanced by its interactions with γ(β_H+γ)(β_H+β_L+γ) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.     

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30?mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30?mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.     

Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T))

Deinococcus proteolyticus (ex Kobatake et al. 1973) Brook and Murray 1981 is one of currently 47 species in the genus Deinococcus within the family Deinococcaceae. Strain MRP(T) was isolated from feces of Lama glama and possesses extreme radiation resistance, a trait is shares with various other species of the genus Deinococcus, with D. proteolyticus being resistant up to 1.5 Mrad of gamma radiation. Strain MRP(T) is of further interest for its carotenoid pigment. The genome presented here is only the fifth completed genome sequence of a member of the genus Deinococcus (and the forth type strain) to be published, and will hopefully contribute to a better understanding of how members of this genus adapted to high gamma- or UV ionizing-radiation. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,886,836 bp long genome with its four large plasmids of lengths 97 kbp, 132 kbp, 196 kbp and 315 kbp harbors 2,741 protein-coding and 58 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Synergistic control of CO2 emissions by fish and nutrients in a humic tropical lake

Using experimental mesocosms, we tested the strength of bottom–up controls by nutrients and top–down controls by an omnivorous fish (Hyphessobrycon bifasciatus; family Characidae), and the interaction between them on the CO₂ partial pressure (pCO₂) in the surface waters of a tropical humic lake (Lake Cabiúnas, Brazil). The experiment included the addition of nutrients and fish to the mesocosms in a factorial design. Overall, persistent CO₂ emissions to the atmosphere, supported by an intense net heterotrophy, were observed in all treatments and replicates over the 6-week study period. The CO₂ efflux (average ± standard error) integrated over the experiment was similar among the control mesocosms and those receiving only fish or only nutrients (309 ± 2, 303 ± 16, and 297 ± 17 mmol CO₂ m⁻² day⁻¹, respectively). However, the addition of nutrients in the presence of fish resulted in a high algal biomass and daytime net autotrophy, reducing the CO₂ emissions by 35% (by 193 ± 7 mmol CO₂ m⁻² day⁻¹). These results indicate that high CO₂ emissions persist following the eutrophication of humic waters, but that the magnitude of these emissions might depend on the structure of the food web. In conclusion, fish and nutrients may act in a synergistic manner to modulate persistent CO₂ emissions from tropical humic lakes.