Supporting Biomedical Research Training for Historically Underrepresented Undergraduates Using Interprofessional, Nonformal Education Structures.

Research experience provides critical training for new biomedical research scientists. Students from underrepresented populations studying science, technology, engineering, and mathematics (STEM) are increasingly recruited into research pathways to diversify STEM fields. However, support structures outside of research settings designed to help these students navigate biomedical research pathways are not always available; nor are program support components outside the context of laboratory technical skills training and formal mentorship well understood. This study leveraged a multi-institutional research training program, Enhancing Cross-Disciplinary Infrastructure and Training at Oregon (EXITO), to explore how nine institutions designed a new curricular structure (Enrichment) to meet a common goal of enhancing undergraduate research training and student success. EXITO undergraduates participated in a comprehensive, 3-year research training program with the Enrichment component offered across nine sites: three universities and six community colleges, highly diverse in size, demographics, and location. Sites' approaches to supporting students in the training program were studied over a 30-month period. All sites independently created their own nonformal curricular structures, implemented interprofessionally via facilitated peer groups. Site data describing design and implementation were thematically coded to identify essential programmatic components across sites, with student feedback used to triangulate findings. Enrichment offered students time to critically reflect on their interests, experiences, and identities in research; network with peers and professionals; and support negotiation of hidden and implicit curricula. Students reported the low-pressure setting and student-centered curriculum balanced the high demands associated with academics and research. Core curricular themes described Enrichment as fostering a sense of community among students, exposing students to career paths and skills, and supporting development of students' professional identities. The non-formal, interprofessional curricula enabled students to model diverse biomedical identities and pathways for each other while informing institutional structures to improve diverse undergraduate students' success in academia and research.

Microbial communities in dark oligotrophic volcanic ice cave ecosystems of Mt. Erebus, Antarctica.

The Earth's crust hosts a subsurface, dark, and oligotrophic biosphere that is poorly understood in terms of the energy supporting its biomass production and impact on food webs at the Earth's surface. Dark oligotrophic volcanic ecosystems (DOVEs) are good environments for investigations of life in the absence of sunlight as they are poor in organics, rich in chemical reactants and well known for chemical exchange with Earth's surface systems. Ice caves near the summit of Mt. Erebus (Antarctica) offer DOVEs in a polar alpine environment that is starved in organics and with oxygenated hydrothermal circulation in highly reducing host rock. We surveyed the microbial communities using PCR, cloning, sequencing and analysis of the small subunit (16S) ribosomal and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RubisCO) genes in sediment samples from three different caves, two that are completely dark and one that receives snow-filtered sunlight seasonally. The microbial communities in all three caves are composed primarily of Bacteria and fungi; Archaea were not detected. The bacterial communities from these ice caves display low phylogenetic diversity, but with a remarkable diversity of RubisCO genes including new deeply branching Form I clades, implicating the Calvin-Benson-Bassham (CBB) cycle as a pathway of CO2 fixation. The microbial communities in one of the dark caves, Warren Cave, which has a remarkably low phylogenetic diversity, were analyzed in more detail to gain a possible perspective on the energetic basis of the microbial ecosystem in the cave. Atmospheric carbon (CO2 and CO), including from volcanic emissions, likely supplies carbon and/or some of the energy requirements of chemoautotrophic microbial communities in Warren Cave and probably other Mt. Erebus ice caves. Our work casts a first glimpse at Mt. Erebus ice caves as natural laboratories for exploring carbon, energy and nutrient sources in the subsurface biosphere and the nutritional limits on life.

Oxidative remobilization of technetium sequestered by sulfide-transformed nano zerovalent iron.

Our previous study showed that formation of TcS2-like phases is favored over TcO2 under sulfidic conditions stimulated by nano zerovalent iron. This study further investigates the stability of Tc(IV) sulfide upon reoxidation by solution chemistry, solid phase characterization, and X-ray absorption spectroscopy. Tc dissolution data showed that Tc(VII) reduced by sulfide-transformed nZVI has substantially slower reoxidation kinetics than Tc(VII) reduced by nZVI only. The initial inhibition of Tc(IV) dissolution at S/Fe = 0.112 is due to the redox buffer capacity of FeS, which is evidenced by the parallel trends in oxidation-reduction potentials (ORP) and Tc dissolution kinetics. The role of FeS in inhibiting Tc oxidation is further supported by the Mössbauer spectroscopy and micro X-ray diffraction data at S/Fe = 0.112, showing persistence of FeS after 24-h oxidation but complete oxidation after 120-h oxidation. X-ray absorption spectroscopy data for S/Fe = 0.011 showed significantly increasing percentages of TcS2 in the solid phase after 24-h oxidation, indicating stronger resistance of TcS2 to oxidation. At S/Fe = 0.112, the XAS results revealed significant transformation of Tc speciation from TcS2 to TcO2 after 120-h oxidation. Given that no apparent Tc dissolution occurred during this period, the speciation transformation might play a secondary role in hindering Tc oxidation. Collectively, the results indicate that sequestrating Tc as TcS2 under stimulated sulfate reduction is a promising strategy to improve the long-term stability of reduced Tc in subsurface remediation.

Reductive sequestration of pertechnetate (99TcO4⁻) by nano zerovalent iron (nZVI) transformed by abiotic sulfide.

Under anoxic conditions, soluble pertechnetate (99TcO4⁻) can be reduced to less soluble TcO2·nH2O, but the oxide is highly susceptible to reoxidation. Here we investigate an alternative strategy for remediation of Tc-contaminated groundwater whereby sequestration as Tc sulfide is favored by sulfidic conditions stimulated by nano zerovalent iron (nZVI). nZVI was pre-exposed to increasing concentrations of sulfide in simulated Hanford groundwater for 24 h to mimic the onset of aquifer biotic sulfate reduction. Solid-phase characterizations of the sulfidated nZVI confirmed the formation of nanocrystalline FeS phases, but higher S/Fe ratios (>0.112) did not result in the formation of significantly more FeS. The kinetics of Tc sequestration by these materials showed faster Tc removal rates with increasing S/Fe between 0 and 0.056, but decreasing Tc removal rates with S/Fe > 0.224. The more favorable Tc removal kinetics at low S/Fe could be due to a higher affinity of TcO4⁻ for FeS than iron oxides, and electron microscopy confirmed that the majority of the Tc was associated with FeS phases. The inhibition of Tc removal at high S/Fe appears to have been caused by excess HS(-). X-ray absorption spectroscopy revealed that as S/Fe increased, the pathway for Tc(IV) formation shifted from TcO2·nH22 to Tc sulfide phases. The most substantial change of Tc speciation occurred at low S/Fe, coinciding with the rapid increase in Tc removal rate. This agreement further confirms the importance of FeS in Tc sequestration.

The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial cells in soil samples without interference from background fluorescence.

Visualization of microorganisms in soils and sediments using fluorescent dyes is a common method in microbial ecology studies, but is often hampered by strong nonspecific background fluorescence that can mask genuine cellular signals. The cyanine nucleic acid binding dyes TO-PRO-3 and TOTO-3 iodide enabled a clear detection of microbial cells in a mineral soil, while nonspecific background was greatly reduced compared with commonly used dyes. When used as counterstains for fluorescence in situ hybridization (FISH), both cyanine dyes allowed identification of microbial cells despite strong background from nonspecifically bound probes. TO-PRO-3 and TOTO-3 are easy to use and represent superior alternatives for detecting microorganisms in soil environments.

Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes.

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNA(Trp). In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNA(Glu). Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TpsiC loop of tRNA(Leu(UUR)). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92-96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.

Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency.

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.

A culture-independent survey of the bacterial community in a radon hot spring.

Paralana is an active, radon-containing hot spring situated in a region of South Australia's Flinders Ranges with a long history of hydrothermal activity. Our aim was to determine the bacterial composition of Paralana using a culture-independent, 16S rRNA-based technique. The presence of a diverse bacterial community was strongly suggested by the large number (approximately 180) of different ribotypes obtained upon analysis of nine hot spring samples. DNA sequencing of Paralana 16S rRNA genes corroborated this observation, identifying representatives of seven confirmed and two candidate divisions of the domain Bacteria. These included Cyanobacteria, Proteobacteria (both beta and delta subdivisions), the Cytophaga-Flexibacter-Bacteroides group, Low G + C Gram-positives, Nitrospira, green non-sulfur bacteria, green sulfur bacteria, OP8, and OP12. No known ionizing radiation-resistant Bacteria were identified. Only one Paralana 16S rRNA sequence type (recombinant B5D) was homologous to a sequence previously identified from a radioactive environment.