Evaluating Natural Source Zone Depletion and Enhanced Source Zone Depletion in laboratory columns via soil redox continuous sensing and microbiome characterization.

To optimally employ Natural Source Zone Depletion (NSZD) and Enhanced Source Zone Depletion (ESZD) at sites impacted by light non-aqueous phase liquids (LNAPL), monitoring strategies are required. Emerging use of subsurface oxidation-reduction potential (ORP) sensors shows promise for tracking redox evolution, which reflects ongoing biogeochemical processes. However, further understanding of how soil redox dynamics relate to subsurface microbial activity and LNAPL degradation pathways is needed. In this work, soil ORP sensors and DNA and RNA sequencing-based microbiome analysis were combined to elucidate NSZD and ESZD (biostimulation via periodic sulfate addition and biosparging) processes in columns containing LNAPL-impacted soils from a former petroleum refinery. Results show expected relationships between continuous soil redox and active microbial communities. Continuous data revealed spatial and temporal detail that informed interpretation of the hydrocarbon biodegradation data. Redox increases were transient for sulfate addition, and sequencing revealed how hydrocarbon concentration and composition impacted microbiome structure and naphthalene degradation. Periodic biosparging did not result in fully aerobic conditions suggesting observed biodegradation improvements could be explained by alternative anaerobic metabolisms (e.g., iron reduction due to air oxidizing reduced iron). Collectively, data suggest combining continuous redox sensing with microbiome analysis provides insights beyond those possible with either monitoring tool alone.

The ISR downstream target ATF4 represses long-term memory in a cell type-specific manner.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

Small nuclear RNAs enhance protein-free RNA-programmable base conversion on mammalian coding transcripts.

Endogenous U small nuclear RNAs (U snRNAs) form RNA-protein complexes responsible for eukaryotic processing of pre-mRNA into mature mRNA. Previous studies have demonstrated the utility of guide-programmable U snRNAs in targeted exon inclusion and exclusion. We investigated whether snRNAs can also enhance conversion of RNA bases over state-of-the-art RNA targeting technologies in human cells. When compared to adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs, we find that guided A>I snRNAs consistently increase adenosine-to-inosine editing efficiency for genes with higher exon counts, perturb substantially fewer genes in the transcriptome, and localize more persistently to the nucleus where ADAR is expressed. A>I snRNAs can also edit pre-mRNA 3' splice sites to promote splicing changes. Finally, snRNA fusions to H/ACA box snoRNAs (U>Ψ snRNAs) increase targeted RNA pseudouridylation efficiency. Altogether, our results advance the protein-free RNA base conversion toolbox and enhance minimally invasive RNA targeting technologies to treat genetic diseases.

Engineered Dual Antioxidant Enzyme Complexes Targeting ICAM-1 on Brain Endothelium Reduce Brain Injury-Associated Neuroinflammation.

The neuroinflammatory cascade triggered by traumatic brain injury (TBI) represents a clinically important point for therapeutic intervention. Neuroinflammation generates oxidative stress in the form of high-energy reactive oxygen and nitrogen species, which are key mediators of TBI pathology. The role of the blood-brain barrier (BBB) is essential for proper neuronal function and is vulnerable to oxidative stress. Results herein explore the notion that attenuating oxidative stress at the vasculature after TBI may result in improved BBB integrity and neuroprotection. Utilizing amino-chemistry, a biological construct (designated "dual conjugate" for short) was generated by covalently binding two antioxidant enzymes (superoxide dismutase 1 (SOD-1) and catalase (CAT)) to antibodies specific for ICAM-1. Bioengineering of the conjugate preserved its targeting and enzymatic functions, as evaluated by real-time bioenergetic measurements (via the Seahorse-XF platform), in brain endothelial cells exposed to increasing concentrations of hydrogen peroxide or a superoxide anion donor. Results showed that the dual conjugate effectively mitigated the mitochondrial stress due to oxidative damage. Furthermore, dual conjugate administration also improved BBB and endothelial protection under oxidative insult in an in vitro model of TBI utilizing a software-controlled stretching device that induces a 20% in mechanical strain on the endothelial cells. Additionally, the dual conjugate was also effective in reducing indices of neuroinflammation in a controlled cortical impact (CCI)-TBI animal model. Thus, these studies provide proof of concept that targeted dual antioxidant biologicals may offer a means to regulate oxidative stress-associated cellular damage during neurotrauma.

Distribution and release of PFAS from AFFF-impacted asphalt: How does it compare to concrete?

Aqueous film forming foam (AFFF)-impacted asphalt and concrete may serve as potential secondary sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surficial leaching. We aimed to understand the vertical distribution and surficial release of PFAS from AFFF-impacted asphalt and concrete cores collected from various locations (∼10-70 m distance between samples). Among the PFAS analyzed, 6:2 FTS was observed as having the highest concentration in the surface layer (0 - 0.5 cm) of concrete (225 µg kg-1) and in the runoff from the concrete (2600 ng L-1). PFOS was detected at the highest concentration in the surface layer (0 - 0.5 cm) of asphalt (47 µg kg-1) and associated runoff (780 ng L-1). The total mass of PFAS released during three rainfall simulations accounts for a fraction of the total mass in the surface layer (0 - 0.5 cm), ranging from 0.10 - 9.8% and 0.078 - 2.4% for asphalt and concrete cores, respectively. Asphalt exhibited a higher release rate than concrete, demonstrated by the higher total release coefficient of PFAS (4 - 16 m-2) compared to that of concrete cores (1 - 5 m-2). These results suggested that, similar to concrete, AFFF-impacted asphalt may be a secondary source of PFAS to the environment.

Insights gained from sequencing Australian non-invasive and invasive Streptococcus pyogenes isolates.

Epidemiological data have indicated that invasive infections caused by the Gram-positive cocci Streptococcus pyogenes (group A streptococcus, GAS) have increased in many Australian states over the past two decades. In July 2022, invasive GAS (iGAS) infections became nationally notifiable in Australia via public-health agencies. Surveillance for S. pyogenes infections has been sporadic within the state of New South Wales (NSW). This has led to a lack of genetic data on GAS strains in circulation, particularly for non-invasive infections, which are the leading cause of GAS's burden on the Australian healthcare system. To address this gap, we used whole-genome sequencing to analyse the genomes of 318 S. pyogenes isolates collected within two geographical regions of NSW. Invasive isolates were collected in 2007-2017, whilst non-invasive isolates were collected in 2019-2021. We found that at least 66 different emm-types were associated with clinical disease within NSW. There was no evidence of any Australian-specific clones in circulation. The M1UK variant of the emm1 global pandemic clone (M1global) has been detected in our isolates from 2013 onwards. We detected antimicrobial-resistance genes (mainly tetM, ermA or ermB genes) in less than 10 % of our 318 isolates, which were more commonly associated with non-invasive infections. Superantigen virulence gene carriage was reasonably proportionate between non-invasive and invasive infection isolates. Our study adds rich data on the genetic makeup of historical S. pyogenes infections within Australia. Ongoing surveillance of invasive and non-invasive GAS infections within NSW by whole-genome sequencing is warranted to inform on outbreaks, antimicrobial resistance and vaccine coverage.

Activation of CB2R by synthetic CB2R agonist, PM289, improves brain endothelial barrier properties, decreases inflammatory response and enhances endothelial repair.

The Cannabinoid 2 Receptor (CB2R) has been found to provide immunological modulation in different cell types. More recently, detection of CB2R in the cerebral endothelium suggests a possible role in the resolution of inflammation at the level of the blood-brain-barrier (BBB). Here, the notion that CB2R upregulation in brain endothelial cells could be exploited to promote vascular protection and BBB integrity was evaluated. Targeting and activation of CB2R was accomplished by a novel and highly specific chromenopyrazole based CB2R agonist, PM289. This study demonstrates that CB2R upregulation is induced as early as 8 h in the cortical vasculature in an experimental mouse model of TBI. Unlike CB2R, CB1R was marginally detected and not significantly induced. In the human brain endothelial cell line, hCMEC/D3 cells, similar induction of CB2R was observed upon stimulation with TNFα. Analysis of transendothelial electrical resistance shows that PM289 markedly prevented the barrier-leakiness induced by TNFα. The BBB is also responsible for maintaining an immunological barrier. The five-fold increase in ICAM1 expression in stimulated endothelial cells was significantly diminished due to CB2R activation. Utilizing wounding assays, results showed that wound repair could be accomplished in nearly half the time when the novel CB2R agonist is present compared to the untreated control. Lastly, mechanistically, the effects of CB2R may be explained by the observed inhibition of the p65 NFκB subunit. Overall, these studies support the notion that targeting and activating CB2R in the brain vasculature could aid in BBB and vascular protection in the context of neuroinflammation.