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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

16S rDNA↗
DOE OSTI2023

Propidium Monoazide (PMAxx)-Recombinase Polymerase Amplification Exo (RPA Exo) Assay for Rapid Detection of Burkholderia cepacia Complex in Chlorhexidine Gluconate (CHX) and Benzalkonium Chloride (BZK) Solutions

Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer–probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 10 4 colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50–500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4–459.3 RFU) and total cells (207.82–684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path

Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3'-5' exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

16S rRNA gene sequencing↗
DOE OSTI2020

An outbreak of Septoria canker caused by Sphaerulina musiva on Populus trichocarpa in eastern Oregon

Branch and stem cankers were observed in a Populus trichocarpa L. plantation in late summer of 2018 near Boardman, Oregon USA. Stem cankers were flat faced with swollen margins and the bark in the center of the cankers was black and depressed (Figure 1). 254 out of 1054 trees (25%) observed had cankers. Symptomatic stems and branches from several trees were brought to Oregon State University for identification. Cankers were surface-disinfested in a 5% NaOCl for 2 min and rinsed twice in deionized H 2 O for 2 min. Bark was removed from cankers and pieces of wood at the margin between healthy and necrotic tissue were plated on KV8 medium amended with streptomycin sulfate at 100 mg liter–1 and chloramphenicol (Amresco) at 240 mg liter–1 (Dunnell and LeBoldus 2017). Eight sporulating colonies typical of Sphaerulina musiva (Peck) Quaedvlieg, Verkley, and Crous (Syn. = Septoria musiva Peck) developed within 7 days of plating. S. musiva was identified by morphological characteristics (conidium size = 28–54 × 3.5–4 µm) and confirmed by comparing the sequence of the ITS region (accession numbers: MN275180-MN275187) to JX901814 with 99% identity. We tested 13 additional cankers collected from the plantation using S. musiva specific primers (Abraham et al. 2018). Eight of the 13 samples were positive for S. musiva DNA. Dormant cuttings from three genotypes of P. trichocarpa (BESC-184; BESC-259; BESC-388) were collected in a field planting near Oregon State University (OSU). These were planted in cone-tainers in the greenhouse at OSU and grown to approximately 30 cm in height. Four replicates of each of the three genotypes were inoculated in the greenhouse with one Oregon isolate using a suspension of 1x10 6 conidia ml-1. The entire tree was sprayed until runoff using a spray bottle (LeBoldus et al. 2010). The experimental design was a completely randomized design with four replicates of each genotype. Sterile deionized H 2 O was sprayed on one control tree of each genotype. The mean number of resulting cankers were BESC-184 = 11, BESC-259 = 1, and BESC-388 = 13.75. The controls did not develop any cankers. The pathogen was successfully reisolated from one canker from each genotype. Sphaerulina musiva can cause severely damaging leaf spot and stem canker diseases in the eastern and central USA and Canada (Bier 1939; Herath et al. 2016). Susceptible Populus species and hybrids develop cankers which can girdle and weaken stems, leading to breakage or tree death (Bier 1939). A previous survey of the region did not detect S. musiva (Newcombe et al. 1995). This outbreak of Septoria canker underscores the potential threat of this pathogen to P. trichocarpa in the Pacific Northwest where it is a keystone species in riparian ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2012

Molecular Technique to Understand Deep Microbial Diversity

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Vaishampayan, Parag A.↗
NASA NTRS2006

Bacterial Composition and Quantitation in the Yungay Region of the Atacama Desert, Chile

Voyager would have produced similar negative results if tested in the Yungay region (Navarro-Gonzales et al, 2003). However, the number of bacteria present per gr'am in the Yungay sands is not well-established, being below the detection limit of culture methods. For the past several years, this group has been analyzing Yungay sands by quantitative Polymerase Chain Reaction (qPCR), both in the lab and in the field, to establish protocols for counting bacteria. We use primers that detect a broad range of bacterial taxa, including both proteobacteria and firmicutes. Optimization of DNA extraction and qPCR analysis is critical for accurate counts. Comparison of qPCR results with PhosphoLipid Fatty Acid (PLFA)-analysis and other counting methods indicates that there are detectable bacteria in sand obtained from the upper few centimeters of the desert surface.

Fletcher, Lauren↗
NASA NTRS2003

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

Evaluation Studies↗
NASA NTRS2003

Autotrophic Ecosystems on the Early Earth

Ophiolite sequences, sections of lower oceanic crust and upper mantle that have been thrust onto continental craton, are located in northern and central California and provide easily accessible areas that serve as good analogs for similar, more extensive areas of the early Earth. We have begun investigating and characterizing these sites in order to understand better the processes that may be responsible for the water chemistry, mineralogy and biology of similar environments on the early Earth. The geophysical and geochemical processes in these terranes provide niches for unique communities of extremeophiles and likely provide a good analog to the location that first gave rise to life on Earth. The ophiolites found in northern and central California include the Trinity, Josephine, Coast Range and Point Sal, all of which are approximately 160 million years old. Fluids from serpentinizing springs are generally alkaline with high pH and H2 contents, indicating that the mafic rock compositions control the fluid composition through water-rock reactions during relatively low-grade hydrothermal processes. There are significant amounts of primary mineralogy remaining in the rocks, meaning that substantial alteration processes are still occurring in these terranes. The general reaction for serpentinization of olivine is given by one of the authors. olivine + H2O = serpentine + brucite + magnetite + H2. We have analyzed the mineralogical composition of several rock samples collected from the Coast Range Ophiolite near Clear Lake, CA by electron microprobe. The remnant primary mineralogy is fairly urnform in composition, with an olivine composition of Fo(sub 90), and with pyroxene compositions of En(sub 90) for orthopyroxene and En(sub 49)Wo(sub 48)Fs(sub 03) for the clinopyroxene. Other primary phases observed include chromites and other spinels. Examination of petrographic thin sections reveals that serpentinization reactions have occurred in these locations. The serpentine resulting from aqueous alteration of olivine resides in veins that are see to cross cut the primary mineral grains. There are several generations of alteration products, comprised mostly of serpentines that are magnesium rich, with magnetite, brucite and carbonates observed as accessory minerals. The formation of carbonates can be taken to indicate the presence of CO2 in the altering fluids. We collected samples from a spring in the Coast Range Ophiolite in order to determine whether the geochemical environment serves as a habitat for chemotrophic microorganisms. DNA was extracted from the sediment samples and the 16s rRNA gene was PCR amplified using universal Archaeal primers. Denaturing gradient gel electrophoresis (DGGE) was used to determine the community of Archaea thriving in these samples. Our results indicate that there were 8 different genera of Archaea from a single sample. A sequence was obtained from one of these eight. The sequence is of an organism similar to Halorubrum tibetense, and alkalophilic Archaeon. This result suggests that these environments are likely hosts for communities of organisms that are adapted for the unique chemistry provided by the alkaline spring.

Schulte, M.↗
NASA NTRS1992

On the early emergence of reverse transcription: theoretical basis and experimental evidence

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.

Non-NASA Center↗
DOE OSTI2022

Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining

DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ’s approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

NASA Discipline Regulatory Physiology↗
NASA NTRS2013

Characterization of Microbial Communities Found in Bioreactor Effluent

The purpose of this investigation was to examine microbial communities of simulated wastewater effluent from hollow fiber membrane bioreactors collected from the Space Life Science Laboratory and Texas Technical University. Microbes were characterized using quantitative polymerase chain reaction where a total count of bacteria and fungi were determined. The primers that were used to determine the total count of bacteria and fungi were targeted for 16S rDNA genes and the internal transcribed spacer, respectively. PCR products were detected with SYBR Green I fluorescent dye and a melting curve analysis was performed to identify unique melt profiles resulting from DNA sequence variations from each species of the community. Results from both the total bacteria and total fungi count assays showed that distinct populations were present in isolates from these bioreactors. This was exhibited by variation in the number of peaks observed on the melting curve analysis graph. Further analysis of these results using species-specific primers will shed light on exactly which microbes are present in these effluents. Information gained from this study will enable the design of a system that can efficiently monitor microbes that play a role in the biogeochemical cycling of nitrogen in wastewater on the International Space Station to assist in the design of a sustainable system capable of converting this nutrient.

Flowe, Candice↗
NASA NTRSDate not supplied

Method Development for Multiplex, In-Situ, and Real-Time Detection of Herpesvirus Reactivation in Spaceflight Crews using Nanopore Sequencing

Reactivation of latent herpesviruses in crews onboard the International Space Station (ISS) is a well-established spaceflight-associated phenomenon and has been linked to overall immune stress. Beyond an indicator of an altered immune state, this stress-induced reactivation of viruses such as herpesvirus simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and Varicella-Zoster virus (VZV) may cause clinical symptoms in crew. There is currently no established protocol for in-flight monitoring, and samples are analyzed post-flight using ground-based assays. A real-time, in-flight method for herpesvirus detection followed by stress-mitigation strategies would be a significant advance. In this study, we developed a real-time assay for the multiplex detection of HSV-1, EBV, and VZV from saliva that could be implemented for in-situ monitoring of ISS crew. This method builds on previously validated spaceflight-compatible portable molecular protocols and platforms, such as the miniPCR thermal cycler and the MinION sequencer. Since a metagenomic approach is not currently permitted for crew samples (NASA policy), we employed multiplexing-ready primers directed toward targeted regions within the HSV-1, EBV, and VZV genomes. We also used primers for the human gene, Statherin (STATH), as an internal control. These primers were validated using spiked-in, positive control HSV-1, EBV, and VZV from virus-negative saliva extracted using the Zymo-Research Quick-DNA/RNA Viral MagBead Kit. The PCR Barcoding Kit was used to prepare DNA libraries that were then sequenced on the MK1C and analyzed against known reference genomes using minimap2. Following validation of this method with spiked saliva samples, suspected herpesvirus-positive clinical saliva samples were successfully tested. Prior to use onboard the ISS, this method will be deployed to an analog environment during overwintering at Palmer Station, Antarctica in 2023. This work represents the successful application of molecular technologies currently onboard the ISS for the real-time monitoring of latent herpesvirus reactivation from saliva samples. This assay, in combination with stress-reduction strategies, holds the potential to manage herpesvirus reactivation in ISS crew, thereby improving health outcomes and overall immunity.

Patrick M. Rydzak↗
NASA NTRS1995

Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae

Representatives of the family Methanosarcinaceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Methanosarcinaceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16S rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 +/- 0.541 times more changes than the respective pairs of 16S rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16S rRNA gene. The qualitative similarity of the mcrI and 16S rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published mcrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinaceae do not have distinct mcrII genes.

NASA Discipline Number 52-30↗
NASA NTRS2004

Characterization of Two Microbial Isolates from Andean Lakes in Bolivia

We are currently investigating the biological population present in the highest and least explored perennial lakes on earth in the Bolivian and Chilean Andes, including several volcanic crater lakes of more than 6000 m elevation, in combination of microbiological and molecular biological methods. Our samples were collected in saline lakes of the Laguna Blanca Laguna Verde area in the Bolivian Altiplano and in the Licancabur volcano crater (27 deg. 47 min S/67 deg. 47 min. W) in the ongoing project studying high altitude lakes. The main goal of the project is to look for analogies with Martian paleolakes. These Bolivian lakes can be described as Andean lakes following the classification of Chong. We have attempted to isolate pure cultures and phylogenetically characterize prokaryotes that grew under laboratory conditions. Sediment samples taken from the Licancabur crater lake (LC), Laguna Verde (LV), and Laguna Blanca (LB) were analyzed and cultured using enriched liquid media under both aerobic and anaerobic conditions. All cultures were incubated at room temperature (15 to 20 C) and under light exposure. For the reported isolates, 36 hours incubation were necessary for reaching optimal optical densities to consider them viable cultures. Ten serial dilutions starting from 1% inoculum were required to obtain a suitable enriched cell culture to transfer into solid media. Cultures on solid medium were necessary to verify the formation of colonies in order to isolate pure cultures. Different solid media were prepared using several combinations of both trace minerals and carbohydrates sources in order to fit their nutrient requirements. The microorganisms formed individual colonies on solid media enriched with tryptone, yeast extract and sodium chloride. Cells morphology was studied by optical and electronic microscopy. Rodshape morphologies were observed in most cases. Total bacterial genomic DNA was isolated from 50 ml late-exponential phase culture by using the CTAB miniprep protocol. The 16S rRNA genes were amplified by PCR using both Bacteria- and Archaeauniversal primer sets: 27f and 1492r, 21f and 1492r respectively. Sequences of 16S rRNA gene were determined and initially compared with reference sequences contained in the EMBL nucleotide sequence database by using the BLAST program and were subsequently aligned with 16S rRNA reference sequences in the ARB package (http://www.mikro.biologie.tu-muenchen.de). Aligned sequences were inserted within a stable phylogenetic tree by using the ARB parsimony tool. In this work we report the morphology and phylogenetic characterization of two isolates belonged to Laguna Blanca sediments.

Demergasso, C.↗
NASA NTRS1998

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Non-NASA Center↗