Aberrant protein folding leading to the formation of characteristic cross-β-sheet-rich amyloid structures is well known for its association with a variety of debilitating human diseases. Often, depending upon amino acid composition, only a small segment of a large protein participates in amyloid formation and is in fact capable of self-assembling into amyloid, independent of the rest of the protein. Therefore, such peptide fragments serve as useful model systems for understanding the process of amyloid formation. An important factor that has often been overlooked while using peptides to mimic full-length protein is the charge on the termini of these peptides. Here, we show the influence of terminal charges on the aggregation of an amyloidogenic peptide from microtubule-associated protein Tau, implicated in Alzheimer's disease and tauopathies. We found that modification of terminal charges by capping the peptide at one or both of the termini drastically modulates the fibrillation of the hexapeptide sequence paired helical filament 6 (PHF6) from repeat 3 of Tau, both with and without heparin. Without heparin, the PHF6 peptide capped at both termini and PHF6 capped only at the N-terminus self-assembled to form amyloid fibrils. With heparin, all capping variants of PHF6, except for PHF6 with both termini free, formed typical amyloid fibrils. However, the rate and extent of aggregation both with and without heparin as well as the morphology of aggregates were found to be highly dependent on the terminal charges. Our molecular dynamics simulations on PHF6 capping variants corroborated our experiments and provided critical insights into the mechanism of PHF6 selfassembly. Overall, our results emphasize the importance of terminal modifications in fibrillation of small peptide fragments and provide significant insights into the aggregation of a small Tau fragment, which is considered essential for Tau filament assembly.

Clean-up of vinyl chloride (VC)-contaminated groundwater could be enhanced by stimulating aerobic VC-oxidizing bacterial populations (e.g., methanotrophs) with amendments such as molecular oxygen. In addition, ethene gas injection could further stimulate a different group of aerobic ethene- and VC-oxidizing bacteria called "etheneotrophs." We estimated the abundance and activity of these different VC-oxidizing bacteria in portions of a dilute groundwater VC plume subjected to oxygen and ethene biostimulation. Pyrosequencing of 16S rRNA genes, amplified from community DNA extracted from five groundwater monitoring wells, revealed that Proteobacteria dominated the microbial community. Among the Proteobacteria, methanotroph relative abundance was 6.00 % (well RB52I), 2.81 % (well RB46D), 56.3 % (well RB58I), 23.8 % (well RB63I), and 2.57 % (well RB64I). Reverse transcription qPCR (RT-qPCR) analysis was used to determined methanotroph and etheneotroph functional gene expression from selected monitoring wells. Resulting transcript per gene ratios for methanotroph functional genes (pmoA and mmoX) were 0.013 (RB46D), 0.017 (RB63I), 0.112 (RB64I), and 0.004 (RB46D), 0.239 (RB63I), and 0.199 (RB64I), respectively. Transcript per gene ratios for etheneotroph functional genes (etnC and etnE) were 0.37 (RB46D), 0.81 (RB63I), 5.85 (RB64I), and 0.38 (RB46D), 0.67 (RB63I), and 2.28 (RB64I), respectively. When considered along with geochemical and contaminant data from these wells, our RT-qPCR results suggest that methanotrophs and etheneotrophs were participating in VC cometabolism. We conclude that these molecular diagnostic techniques could be helpful to site managers interested in documenting the effectiveness of VC bioremediation strategies.

Bioremediation of vinyl chloride (VC) contamination in groundwater could be mediated by three major bacterial guilds: anaerobic VC-dechlorinators, methanotrophs, and ethene-oxidizing bacteria (etheneotrophs) via metabolic or cometabolic pathways. We collected 95 groundwater samples across 6 chlorinated ethene-contaminated sites and searched for relationships among VC biodegradation gene abundance and expression and site geochemical parameters (e.g., VC concentrations). Functional genes from the three major VC-degrading bacterial guilds were present in 99% and expressed in 59% of the samples. Etheneotroph and methanotroph functional gene abundances ranged from 10 to 10 genes per liter of groundwater among the samples with VC reductive dehalogenase gene (bvcA and vcrA) abundances reaching 10 genes per liter of groundwater. Etheneotroph functional genes (etnC and etnE) and VC reductive dehalogenase genes (bvcA and vcrA) were strongly related to VC concentrations (p < 0.001). Methanotroph functional genes (mmoX and pmoA) were not related to VC concentration (p > 0.05). Samples from sites with bulk VC attenuation rates >0.08 year contained higher levels of etheneotroph and anaerobic VC-dechlorinator functional genes and transcripts than those with bulk VC attenuation rates <0.004 year. We conclude that both etheneotrophs and anaerobic VC-dechlorinators have the potential to simultaneously contribute to VC biodegradation at these sites.

Vinyl chloride (VC), a known human carcinogen, is a common and persistent groundwater pollutant at many chlorinated solvent contaminated sites. The remediation of such sites is challenging because of the lack of knowledge on the microorganisms responsible for in situ VC degradation. To address this, the microorganisms involved in carbon assimilation from VC were investigated in a culture enriched from contaminated site groundwater using stable isotope probing (SIP) and high-throughput sequencing. The mixed culture was added to aerobic media, and these were amended with labeled ((13)C-VC) or unlabeled VC ((12)C-VC). The cultures were sacrificed on days 15, 32, and 45 for DNA extraction. DNA extracts and SIP ultracentrifugation fractions were subject to sequencing as well as quantitative PCR (qPCR) for a functional gene linked to VC-assimilation (etnE). The gene etnE encodes for epoxyalkane coenzyme M transferase, a critical enzyme in the pathway for VC degradation. The relative abundance of phylotypes was compared across ultracentrifugation fractions obtained from the (13)C-VC- and (12)C-VC-amended cultures. Four phylotypes were more abundant in the heavy fractions (those of greater buoyant density) from the (13)C-VC-amended cultures compared to those from the (12)C-VC-amended cultures, including Nocardioides, Brevundimonas, Tissierella, and Rhodoferax. Therefore, both a previously identified VC-assimilating genus (Nocardioides) and novel microorganisms were responsible for carbon uptake. Enrichment of etnE with time was observed in the heavy fractions, and etnE sequences illustrated that VC-assimilators harbor similar Nocardioides-like etnE. This research provides novel data on the microorganisms able to assimilate carbon from VC.

Vinyl chloride (VC) is a frequent groundwater contaminant and a known human carcinogen. Bioremediation is a potential cleanup strategy for contaminated sites; however, little is known about the bacteria responsible for aerobic VC degradation in mixed microbial communities. In attempts to address this knowledge gap, the microorganisms able to assimilate labeled carbon ((13)C) from VC within a mixed culture capable of rapid VC degradation (120 μmol in 7 days) were identified using stable isotope probing (SIP). For this, at two time points during VC degradation (days 3 and 7), DNA was extracted from replicate cultures initially supplied with labeled or unlabeled VC. The extracted DNA was ultracentrifuged, fractioned, and the fractions of greater buoyant density (heavy fractions, 1.758 to 1.780 g mL(-1)) were subject to high-throughput sequencing. Following this, specific primers were designed for the most abundant phylotypes in the heavy fractions. Then, quantitative PCR (qPCR) was used across the buoyant density gradient to confirm label uptake by these phylotypes. From qPCR and/or sequencing data, five phylotypes were found to be dominant in the heavy fractions, including Nocardioides (∼40 %), Sediminibacterium (∼25 %), Aquabacterium (∼17 %), Variovorax (∼6 %), and Pseudomonas (∼1 %). The abundance of two functional genes (etnC and etnE) associated with VC degradation was also investigated in the SIP fractions. Peak shifts of etnC and etnE gene abundance toward heavier fractions were observed, indicating uptake of (13)C into the microorganisms harboring these genes. Analysis of the total microbial community indicated a significant dominance of Nocardioides over the other label-enriched phylotypes. Overall, the data indicate Nocardioides is primarily responsible for VC degradation in this mixed culture, with the other putative VC degraders generating a small growth benefit from VC degradation. The specific primers designed toward the putative VC degraders may be of use for investigating VC degradation potential at contaminated sites.

Epoxyalkane:coenzyme M transferase (EaCoMT) plays a critical role in the aerobic biodegradation and assimilation of alkenes, including ethene, propene, and the toxic chloroethene vinyl chloride (VC). To improve our understanding of the diversity and distribution of EaCoMT genes in the environment, novel EaCoMT-specific terminal-restriction fragment length polymorphism (T-RFLP) and nested-PCR methods were developed and applied to groundwater samples from six different contaminated sites. T-RFLP analysis revealed 192 different EaCoMT T-RFs. Using clone libraries, we retrieved 139 EaCoMT gene sequences from these samples. Phylogenetic analysis revealed that a majority of the sequences (78.4%) grouped with EaCoMT genes found in VC-and ethene-assimilating Mycobacterium strains and Nocardioides sp. strain JS614. The four most-abundant T-RFs were also matched with EaCoMT clone sequences related to Mycobacterium and Nocardioides strains. The remaining EaCoMT sequences clustered within two emergent EaCoMT gene subgroups represented by sequences found in propene-assimilating Gordonia rubripertincta strain B-276 and Xanthobacter autotrophicus strain Py2. EaCoMT gene abundance was positively correlated with VC and ethene concentrations at the sites studied. IMPORTANCEThe EaCoMT gene plays a critical role in assimilation of short-chain alkenes, such as ethene, VC, and propene. An improved understanding of EaCoMT gene diversity and distribution is significant to the field of bioremediation in several ways. The expansion of the EaCoMT gene database and identification of incorrectly annotated EaCoMT genes currently in the database will facilitate improved design of environmental molecular diagnostic tools and high-throughput sequencing approaches for future bioremediation studies. Our results further suggest that potentially significant aerobic VC degraders in the environment are not well represented in pure culture. Future research should aim to isolate and characterize aerobic VC-degrading bacteria from these underrepresented groups. Short-chain alkenes (e.g., ethene, propene, and butenes) are common hydrocarbons in the environment, primarily encountered as fossil fuel components or products of living organisms or generated by the chemical industry (1). For instance, ethene is generated by both plants (2) and bacteria (3).Chlorinated alkenes (e.g., vinyl chloride [VC]) are also naturally occurring, albeit at very low levels (4). VC is produced industrially as a monomer for polyvinyl chloride plastics. However, most environmental VC is generated by incomplete anaerobic dechlorination of the widely used solvents tetrachloroethene (PCE) and trichloroethene (TCE) in groundwater, where ethene can also be generated as a complete dechlorination product (1). PCE, TCE, and VC are common groundwater contaminants (5), and sites contaminated with chloroethenes are widely distributed across the United States (6) and elsewhere (1, 7). VC is of particular concern as a known human carcinogen (8).In aerobic bacteria that utilize short...

Submerged attached growth bioreactors (SAGBs) were operated at 20 °C for 30 weeks in smart-aerated, partial nitritation ANAMMOX mode and in a timer-controlled, cyclic aeration mode. The smart-aerated SAGBs removed 48-53% of total nitrogen (TN) compared to 45% for SAGBs with timed aeration. Low dissolved oxygen concentrations and cyclic pH patterns in the smart-aerated SAGBs suggested conditions favorable to partial nitritation ANAMMOX and stoichiometrically-derived and numerically modeled estimations attributed 63-68% and 14-44% of TN removal to partial nitritation ANAMMOX in these bioreactors, respectively. Ammonia removals of 36-67% in the smart-aerated SAGBs, with measured oxygen and organic carbon limitations, further suggest partial nitritation ANAMMOX. The smart-aerated SAGBs required substantially less aeration to achieve TN removals similar to SAGBs with timer-controlled aeration. Genomic DNA testing confirmed that the dominant ANAMMOX seed bacteria, received from a treatment plant utilizing the DEMON® sidestream deammonification process, was a Candidatus Brocadia sp. (of the Planctomycetales order). The DNA from these bacteria was also present in the SAGBs at the conclusion of the study providing evidence for attached growth and limited biomass washout.

Amyloid β (Aβ) protein is responsible for Alzheimer's disease, and one of its important fragments, , is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aβ(25−35)'s toxicity in cells even though they share similar primary structures with . Here, we seek to understand the molecular mechanisms of how these peptides interact with Aβ(25−35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aβ(25−35), NK, Kassinin, Substance P, and mixtures of the latter three with Aβ(25−35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of , which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aβ(25−35), NK, Kassinin, and Substance P do not form hexamers with a β-sheet structure like Aβ(25− 35). The hexamer structure of Aβ(25−35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aβ(25−35) are discussed.