Rapid and Highly Selective Fe(IV) Generation by Fe(II)-Peroxyacid Advanced Oxidation Processes: Mechanistic Investigation via Kinetics and Density Functional Theory.

High-valent iron (Fe(IV/V/VI)) has been widely applied in water decontamination. However, common Fe(II)-activating oxidants including hydrogen peroxide (H2O2) and persulfate react slowly with Fe(II) and exhibit low selectivity for Fe(IV) production due to the cogeneration of radicals. Herein, we report peroxyacids (POAs; R-C(O)OOH) that can react with Fe(II) more than 3 orders of magnitude faster than H2O2, with high selectivity for Fe(IV) generation. Rapid degradation of bisphenol A (BPA, an endocrine disruptor) was achieved by the combination of Fe(II) with performic acid (PFA), peracetic acid (PAA), or perpropionic acid (PPA) within one second. Experiments with phenyl methyl sulfoxide (PMSO) and tert-butyl alcohol (TBA) revealed Fe(IV) as the major reactive species in all three Fe(II)-POA systems, with a minor contribution of radicals (i.e., •OH and R-C(O)O•). To understand the exceptionally high reactivity of POAs, a detailed computational comparison among the Fenton-like reactions with step-by-step thermodynamic evaluation was conducted. The high reactivity is attributed to the lower energy barriers for O-O bond cleavage, which is determined as the rate-limiting step for the Fenton-like reactions, and the thermodynamically favorable bidentate binding pathway of POA with iron. Overall, this study advances knowledge on POAs as novel Fenton-like reagents and sheds light on computational chemistry for these systems.

Bacteria and Virus Inactivation: Relative Efficacy and Mechanisms of Peroxyacids and Chlor(am)ine.

Peroxyacids (POAs) are a promising alternative to chlorine for reducing the formation of disinfection byproducts. However, their capacity for microbial inactivation and mechanisms of action require further investigation. We evaluated the efficacy of three POAs (performic acid (PFA), peracetic acid (PAA), and perpropionic acid (PPA)) and chlor(am)ine for inactivation of four representative microorganisms (Escherichia coli (Gram-negative bacteria), Staphylococcus epidermidis (Gram-positive bacteria), MS2 bacteriophage (nonenveloped virus), and Φ6 (enveloped virus)) and for reaction rates with biomolecules (amino acids and nucleotides). Bacterial inactivation efficacy (in anaerobic membrane bioreactor (AnMBR) effluent) followed the order of PFA > chlorine > PAA ≈ PPA. Fluorescence microscopic analysis indicated that free chlorine induced surface damage and cell lysis rapidly, whereas POAs led to intracellular oxidative stress through penetrating the intact cell membrane. However, POAs (50 µM) were less effective than chlorine at inactivating viruses, achieving only ∼1-log PFU removal for MS2 and Φ6 after 30 min of reaction in phosphate buffer without genome damage. Results suggest that POAs' unique interaction with bacteria and ineffective viral inactivation could be attributed to their selectivity toward cysteine and methionine through oxygen-transfer reactions and limited reactivity for other biomolecules. These mechanistic insights could inform the application of POAs in water and wastewater treatment.

Isoflurane Disrupts Postsynaptic Density-95 Protein Interactions Causing Neuronal Synapse Loss and Cognitive Impairment in Juvenile Mice via Canonical NO-mediated Protein Kinase-G Signaling.

BACKGROUND: Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein. METHODS: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed. RESULTS: Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1. CONCLUSIONS: Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

First Organoid Intelligence (OI) workshop to form an OI community.

The brain is arguably the most powerful computation system known. It is extremely efficient in processing large amounts of information and can discern signals from noise, adapt, and filter faulty information all while running on only 20 watts of power. The human brain's processing efficiency, progressive learning, and plasticity are unmatched by any computer system. Recent advances in stem cell technology have elevated the field of cell culture to higher levels of complexity, such as the development of three-dimensional (3D) brain organoids that recapitulate human brain functionality better than traditional monolayer cell systems. Organoid Intelligence (OI) aims to harness the innate biological capabilities of brain organoids for biocomputing and synthetic intelligence by interfacing them with computer technology. With the latest strides in stem cell technology, bioengineering, and machine learning, we can explore the ability of brain organoids to compute, and store given information (input), execute a task (output), and study how this affects the structural and functional connections in the organoids themselves. Furthermore, understanding how learning generates and changes patterns of connectivity in organoids can shed light on the early stages of cognition in the human brain. Investigating and understanding these concepts is an enormous, multidisciplinary endeavor that necessitates the engagement of both the scientific community and the public. Thus, on Feb 22-24 of 2022, the Johns Hopkins University held the first Organoid Intelligence Workshop to form an OI Community and to lay out the groundwork for the establishment of OI as a new scientific discipline. The potential of OI to revolutionize computing, neurological research, and drug development was discussed, along with a vision and roadmap for its development over the coming decade.

Evaluating Pain and Analgesia Effectiveness Following Routine Castration in Rabbits Using Behavior and Facial Expressions.

Prevention of pain in rabbits is a priority for both welfare and validity of scientific data. We aimed to determine if the rabbit grimace scale (RbtGS) could be used as a viable, rapid assessment tool in two breeds of rabbit, Dutch belted (DB) and New Zealand white (NZW), following orchidectomy, as an adjunct to behavioral analysis. All animals received analgesia. Rabbits were filmed and their behavior was recorded at multiple time points pre- and post-orchidectomy. Observers then scored specific pain associated behaviors for analysis. Time matched footage was also scored using the rabbit grimace scale (RbtGS). Following surgery, rabbits showed significant increases in the duration spent displaying key pain associated behaviors at 1 and 5 h post-surgery. DB rabbits that received low dose meloxicam (0.2 mg/kg) showed significantly more pain behaviors at 1 and 5 h post-surgery compared to those administered a combination of higher dose meloxicam (0.6 mg/kg) and a lidocaine/bupivacaine local infusion. DB rabbits showed an increase in RbtGS score at both 1 and 5 h post-surgery. In the NZW rabbits, an increase in RbtGS score was only observed at 1 h post-surgery. Using behavioral analysis as the gold standard for comparison, the RbtGS was an effective means of determining when rabbits are painful following orchidectomy. Higher dose meloxicam (0.6 mg/kg) combined with local anesthetic was a more effective method of reducing pain, compared to lower dose meloxicam (0.2 mg/kg) alone.

Methamphetamine-associated cleavage of the synaptic adhesion molecule intercellular adhesion molecule-5.

Methamphetamine (MA) is a highly addictive psychostimulant that, used in excess, may be neurotoxic. Although the mechanisms that underlie its addictive potential are not completely understood, in animal models matrix metalloproteinase (MMP) inhibitors can reduce behavioral correlates of addiction. In addition, evidence from genome-wide association studies suggests that polymorphisms in synaptic cell-adhesion molecules (CAMs), known MMP substrates, are linked to addictive potential in humans. In the present study, we examined the ability of MA to stimulate cleavage of intercellular adhesion molecule-5 (ICAM-5), a synaptic CAM expressed on dendritic spines in the telencephalon. Previous studies have shown that shedding of ICAM-5 is associated with maturation of dendritic spines, and that MMP-dependent shedding occurs with long term potentiation. Herein, we show that MA stimulates ectodomain cleavage of ICAM-5 in vitro, and that this is abrogated by a broad spectrum MMP inhibitor. We also show that an acute dose of MA, administered in vivo, is associated with cleavage of ICAM-5 in murine hippocampus and striatum. This occurs within 6 h and is accompanied by an increase in MMP-9 protein. In related experiments, we examined the potential consequences of ICAM-5 shedding. We demonstrate that the ICAM-5 ectodomain can interact with ß(1) integrins, and that it can stimulate ß(1) integrin-dependent phosphorylation of cofilin, an event that has previously been linked to MMP-dependent spine maturation. Together these data support an emerging appreciation of MMPs as effectors of synaptic plasticity and suggest a mechanism by which MA may influence the same.

Severity and Distribution of Wounds in Rhesus Macaques (Macaca mulatta) Correlate with Observed Self-Injurious Behavior.

Self-injurious behavior (SIB) occurs within laboratory-housed NHP at low frequency but can have a devastating effect on animal research and wellbeing. One barrier to the study and clinical management of these cases is the cost of equipment and personnel time to quantify the behavior according to the current standard of observation and to score remotely obtained video recordings. In studies of human SIB, in which direct observation is difficult or prohibited, researchers have demonstrated that quantifying the tissue damage resulting from SIB can be a useful proxy to represent the underlying behavior. We hypothesized that the nature of wounds resulting from SIB in NHP could be used in a similar manner to measure the abnormal behavior. Using a cohort of rhesus macaques with high-incidence SIB, we examined severity, distribution, and number of wounds and compared them with observed incidences of SIB during a 12-wk experiment. We found that the number, severity, and distribution of physical wounds were associated with the incidences of biting behavior observed during the 2 wk prior to measurement. We also found that an increased number of wounds was associated with increased severity. Animals with wounds of moderate severity were more likely to also have severe wounds than were macaques with wounds that were lower than moderate in severity. This work is the first representative study in NHP to find that behavioral SIB correlates with physical wounding and that increases in the frequency and number of the body regions affected correlates with the severity of wounding.

Use of an aquarium as a novel enrichment item for singly housed rhesus macaques (Macaca mulatta).

Locomotor stereotypies are behaviors often seen in singly housed rhesus macaques (Macaca mulatta) and are considered to represent a maladaptive response to captive environments. Active and passive enrichment items are commonly used to mitigate these and other abnormal behaviors. Active enrichment items allow physical manipulation and may be temporarily successful in reducing stereotypies, but their beneficial effects usually are confined to relatively short periods of active use. Passive enrichment items that do not involve physical manipulation are less well studied, and the results are mixed. This study evaluated an aquarium with live fish for use as a novel passive enrichment item in a common facility setting as a means to decrease locomotor stereotypy. We hypothesized that the introduction of the aquarium would decrease the frequency of locomotor stereotypy in a group of singly housed rhesus macaques (n = 11) with a known history of abnormal behaviors. Unexpectedly, locomotor stereotypy increased with the introduction of the aquarium and then decreased over time. Furthermore, when the aquarium was removed, the frequency of stereotypy decreased to below baseline levels. These unexpected results are best explained by neophobia, a common phenomenon documented in many animal species. The increase in abnormal behavior is likely to result from the addition of a novel object within the environment. This study demonstrates that, in the context of reducing abnormal behavior, presumably innocuous enrichment items may have unexpected effects and should be evaluated critically after their introduction to a captive population.