Adolescent high fat diet alters the transcriptional response of microglia in the prefrontal cortex in response to stressors in both male and female mice.

Both obesity and high fat diets (HFD) have been associated with an increase in inflammatory gene expression within the brain. Microglia play an important role in early cortical development and may be responsive to HFD, particularly during sensitive windows, such as adolescence. We hypothesized that HFD during adolescence would increase proinflammatory gene expression in microglia at baseline and potentiate the microglial stress response. Two stressors were examined, a physiological stressor [lipopolysaccharide (LPS), IP] and a psychological stressor [15 min restraint (RST)]. From 3 to 7 weeks of age, male and female mice were fed standard control diet (SC, 20% energy from fat) or HFD (60% energy from fat). On P49, 1 h before sacrifice, mice were randomly assigned to either stressor exposure or control conditions. Microglia from the frontal cortex were enriched using a Percoll density gradient and isolated via fluorescence-activated cell sorting (FACS), followed by RNA expression analysis of 30 genes (27 target genes, three housekeeping genes) using Fluidigm, a medium throughput qPCR platform. We found that adolescent HFD induced sex-specific transcriptional response in cortical microglia, both at baseline and in response to a stressor. Contrary to our hypothesis, adolescent HFD did not potentiate the transcriptional response to stressors in males, but rather in some cases, resulted in a blunted or absent response to the stressor. This was most apparent in males treated with LPS. However, in females, potentiation of the LPS response was observed for select proinflammatory genes, including Tnfa and Socs3. Further, HFD increased the expression of Itgam, Ikbkb, and Apoe in cortical microglia of both sexes, while adrenergic receptor expression (Adrb1 and Adra2a) was changed in response to stressor exposure with no effect of diet. These data identify classes of genes that are uniquely affected by adolescent exposure to HFD and different stressor modalities in males and females.

Perinatal morphine but not buprenorphine affects gestational and offspring neurobehavioral outcomes in mice.

Within the national opioid epidemic, there has been an increase in the number of infants exposed to opioids in utero. Additionally, opioid agonist medications are the standard of care for women with opioid use disorder during pregnancy. Buprenorphine (BUP), a partial µ -opioid receptor agonist, has been successful in improving gestational and neonatal outcomes. However, in utero exposure has been linked to childhood cognitive and behavioral problems. Therefore, we sought to compare offspring cognitive and behavioral outcomes after prenatal exposure to a clinically relevant low dose of BUP compared to morphine (MO), a full µ -opioid receptor agonist and immediate metabolite of heroin. We used a mouse model to assess gestational and offspring outcomes. Mouse dams were injected once daily s.c. with saline (SAL, n = 12), MO (10 mg/kg, n = 15), or BUP (0.1 mg/kg, n = 16) throughout pre-gestation, gestation, and lactation until offspring were weaned on postnatal day (P)21. Offspring social interaction and exploratory behavior were assessed, along with executive function via the touchscreen 5 choice serial reaction time task (5CSRTT). We then quantified P1 brain gene expression in the frontal cortex and amygdala (AMG). Perinatal MO but not BUP exposure decreased gestational weight gain and was associated with dystocia. In adolescent offspring, perinatal MO but not BUP exposure increased social exploration in males and grooming behavior in females. In the 5CSRTT, male MO exposed offspring exhibited increased impulsive action errors compared to male BUP offspring. In the AMG of P1 MO exposed offspring, we observed an increase in gene expression of targets related to activity of microglia. Importantly, both MO and BUP caused acute hyperlocomotion in the dams to a similar degree, indicating that the selected doses are comparable, in accordance with previous dose comparisons on analgesic and reward efficacy. These data suggest that compared to MO, low dose BUP improves gestational outcomes and has less of an effect on the neonatal offspring brain and later adolescent and adult behavior.

The Safety of Soy Leghemoglobin Protein Preparation Derived from Pichia pastoris Expressing a Soy Leghemoglobin Gene from Glycine max: In Vitro and In Vivo Studies.

Soy leghemoglobin (LegH) protein derived from soy (Glycine max) produced in Pichia pastoris (reclassified as Komagataella phaffii) as LegH Prep is a novel food ingredient that provides meat-like flavor and aroma to plant-derived food products. The safety of LegH Prep has been previously assessed in a battery of in vivo and in vitro testing and found no adverse effects under the conditions tested. In this new work, we present the results of new in vivo and in vitro tests evaluating the safety of LegH Prep. LegH Prep was nonmutagenic in a bacterial reverse mutation assay and nonclastogenic in an in vitro micronucleus assay in human lymphocytes. Systemic toxicity was evaluated in the 90 day dietary study in male and female Sprague-Dawley® rats that included a 28 day recovery period. The study resulted in no animal deaths associated with the administration of LegH Prep at the highest dose (90,000 ppm). There were no significant adverse clinical or physical changes attributed to LegH Prep administration, and no observed adverse effects on either male or female rats over the course of the 28 day recovery phase study. The new 90 day dietary toxicity study established a no observed adverse effect level (NOAEL) of 4798.3 and 5761.5 mg/kg/day, the maximum level tested for male and female rats, respectively. Thus, the results of the studies demonstrate that under the conditions tested, LegH Prep is not toxic for consumption in meat analog products.

Evaluation of the cytotoxicity and genotoxicity of glufosinate-ammonium at technical and commercial grades in HepG2 cells.

Exposure to genotoxic agents is associated with the development of cancer and related diseases. For this reason, assessing the genotoxicity of chemical compounds is necessary. In this line, information about the genotoxic effect of glufosinate-ammonium (GLA) has been reported only for the technical grade. However, humans are frequently exposed to commercial formulations of pesticides. Commercial formulations are characterized by using inner agents that increase toxicity compared to pesticides in technical grade. This study aimed to determine the cytotoxic and genotoxic effects of GLA on HepG2 cells. MTT and comet assays were performed to evaluate cell viability and DNA damage, respectively. HepG2 cells were exposed for 24 h to different concentrations of GLA (at 0.01 µg/mL; 0.04 µg/mL; 0.1 µg/mL; 0.24 µg/mL; 0.52 µg/mL; 1.25 µg/mL; 2.62 µg/mL and 13.12 µg/mL) in commercial- (Finale Ultra®) or technical-grade (GLAT). The results indicated that only Finale Ultra® induced a reduction in cell viability at 13.12 µg/mL. Furthermore, exposure to Finale Ultra® or GLAT was associated with increased DNA damage at concentrations from 0.52-13.12- µg/mL. This study shows the genotoxic effect of GLA on HepG2 cells.

Early life cancer and chemotherapy lead to cognitive deficits related to alterations in microglial-associated gene expression in prefrontal cortex.

Children that survive leukemia are at an increased risk for cognitive difficulties. A better understanding of the neurobiological changes in response to early life chemotherapy will help develop therapeutic strategies to improve quality of life for leukemia survivors. To that end, we used a translationally-relevant mouse model consisting of leukemic cell line (L1210) injection into postnatal day (P)19 mice followed by methotrexate, vincristine, and leucovorin chemotherapy. Beginning one week after the end of chemotherapy, social behavior, recognition memory and executive function (using the 5 choice serial reaction time task (5CSRTT)) were tested in male and female mice. Prefrontal cortex (PFC) and hippocampus (HPC) were collected at the conclusion of behavioral assays for gene expression analysis. Mice exposed to early life cancer + chemotherapy were slower to progress through increasingly difficult stages of the 5CSRTT and showed an increase in premature errors, indicating impulsive action. A cluster of microglial-related genes in the PFC were found to be associated with performance in the 5CSRTT and acquisition of the operant response, and long-term changes in gene expression were evident in both PFC and HPC. This work identifies gene expression changes in PFC and HPC that may underlie cognitive deficits in survivors of early life exposure to cancer + chemotherapy.

Use of non-intrusive laser exfoliation to improve substance uptake into citrus leaves.

Background: Despite the presence of stomata in leaves, foliar application of agrochemicals can be extremely inefficient due to the low permeability of leaf cuticular surfaces to polar compounds. Methods: This study introduced a laser-based "wax exfoliation" method to facilitate the penetration of substances into the leaf and, together with enhancing their uptake into the phloem and subsequent transport across tissue. This investigation demonstrated the effectiveness and non-invasive properties of laser exfoliation to improve the penetration of foliar-applied substances into citrus leaves. Results: This work presents the use of laser energy to exfoliate the cuticle of a leaf, with the highest energy density of 0.76 J/ cm2 resulting in 85-90% exfoliation across the entire laser-spot area. The infrared wavelength of the erbium laser is specifically chosen to target the wax cuticle without causing damage to the underlying epidermal cells. This selective ablation allows for increased penetration of therapeutic compounds into the leaf and transportation throughout the plant's vasculature. This is demonstrated using a fluorescent glucose analog applied to the laser treated leaves, showing increased penetration and transport throughout the leaf. Conclusions: Our findings demonstrate that the use of laser technology for the foliar application of agrochemicals provides significant advantages, including improved foliage uptake of therapeutic compounds. The method of cuticle exfoliation presented in this study is highly effective and non-intrusive, limiting its effects to the cuticle only. Future work should focus on the development of prototypes for in-field applications, including testing at longer distances as the Er:YAG laser does not require a lens for this application.

Perinatal Morphine Exposure Leads to Sex-Dependent Executive Function Deficits and Microglial Changes in Mice.

Children exposed prenatally to opioids are at an increased risk for behavioral problems and executive function deficits. The prefrontal cortex (PFC) and amygdala (AMG) regulate executive function and social behavior and are sensitive to opioids prenatally. Opioids can bind to toll-like receptor 4 (TLR4) to activate microglia, which may be developmentally important for synaptic pruning. Therefore, we tested the effects of perinatal morphine exposure on executive function and social behavior in male and female mouse offspring, along with microglial-related and synaptic-related outcomes. Dams were injected once daily subcutaneously with saline (n = 8) or morphine (MO; 10 mg/kg; n = 12) throughout pregestation, gestation, and lactation until offspring were weaned on postnatal day 21 (P21). Male MO offspring had impairments in attention and accuracy in the five-choice serial reaction time task, while female MO offspring were less affected. Targeted gene expression analysis at P21 in the PFC identified alterations in microglial-related and TLR4-related genes, while immunohistochemical analysis in adult brains indicated decreased microglial Iba1 and phagocytic CD68 proteins in the PFC and AMG in males, but females had an increase. Further, both male and female MO offspring had increased social preference. Overall, these data demonstrate male vulnerability to executive function deficits in response to perinatal opioid exposure and evidence for disruptions in neuron-microglial signaling.

A novel use of the endoscopic cyclophotocoagulative probe for the management of excisional goniotomy induced chronic recurrent hyphema.

Purpose: To describe the management of a case of chronic, recurrent hyphema following excisional goniotomy with the Kahook Dual Blade. Observations: One week following uncomplicated surgery, the patient presented with eye pain, elevated intraocular pressure, and layered hyphema. The hyphema resolved with conservative measures but recurred several times with similar symptoms over the next several months. An attempt to stenose the "oozing" collector channel using the argon laser was unsuccessful. Treatment of the angle with an endoscopic semi-conductor diode laser (endoscopic cyclophotocoagulative or ECP) probe successfully altered the angle preventing further episodes of recurrent hyphema. The primary glaucoma procedure failed necessitating a filtering procedure. Conclusions and importance: Clinician should be aware of this potential complication, its treatment options, and outcomes.

High fat diet consumption restricted to adolescence has minimal effects on adult executive function that vary by sex.

Early life environment can have a lasting effect on brain development and behavior. Diet is a potent environmental factor that can positively or negatively affect neurodevelopment, and unfortunately, the likelihood of a poor diet is high during adolescence. Adverse effects of adolescent high fat diet have been observed on reward-related behaviors, reversal learning, and hippocampal-dependent learning tasks in rodents when tested in adulthood. The prefrontal cortex (PFC) continues to develop throughout adolescence and is thus vulnerable to environmental insults such as poor diet. Therefore, we sought to examine the effects of a high fat diet (HFD) consumed only during adolescence on later life adult PFC-dependent executive function. Male and female mice were fed a HFD (60% energy from fat) during either early or late adolescence then switched to standard chow and tested in a battery of touchscreen-based operant tests of executive function in adulthood. Contrary to our prediction of an adverse effect of HFD, there was no effect of adolescent HFD in males, and females showed faster learning and decreased inattention in adulthood. We conclude that the effects of adolescent-limited HFD on adult executive function are mild, positive, and vary by sex.

Translationally relevant mouse model of early life cancer and chemotherapy exposure results in brain and small intestine cytokine responses: A potential link to cognitive deficits.

Survivors of acute lymphoblastic leukemia (ALL), the most common childhood cancer, are at increased risk for long-term cognitive problems, including executive function deficits. The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients and is closely associated with cognitive deficits. To address how early life cancer chemotherapy leads to cognitive deficits, we developed a translationally relevant mouse model of leukemia survival that exposed mice to leukemic cells and chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) in early life. Male and female mice were tested several weeks later using novel object recognition (recognition memory) and 5-choice serial reaction time task (executive function). Gene expression of proinflammatory, white matter and synapse-associated molecules was assessed in the prefrontal cortex and small intestine both acutely after chemotherapy and chronically after cognitive testing. Early life cancer-chemotherapy exposure resulted in recognition memory and executive function deficits in adult male mice. Prefrontal cortex expression of the chemokine Ccl2 was increased acutely, while small intestine expression of the proinflammatory cytokine tumor necrosis factor-alpha was elevated both acutely (both sexes) and chronically (males only). Inflammation in the small intestine was correlated with prefrontal cortical proinflammatory and synaptic gene expression changes, as well as to executive function deficits. Collectively, these data indicate that the current protocol results in a robust mouse model in which to study cognitive deficits in leukemia survivors, and suggest that small intestine inflammation may represent a novel contributor to adverse CNS consequences of early life chemotherapy.

Neural functions of small heat shock proteins.

Stress response is a cellular widespread mechanism encoded by a common protein program composed by multiple cellular factors that converge in a defense reaction to protect the cell against damage. Among many mechanisms described, heat shock proteins were proposed as universally conserved protective factors in the stress core proteome, coping with different stress stimuli through its canonical role in protein homeostasis. However, emerging evidences reveal non-canonical roles of heat shock proteins relevant for physiological and pathological conditions. Here, we review the implications of inducible heat shock proteins in the central nervous system physiology. In particular, we discuss the relevance of heat shock proteins in the maintenance of synapses, as a balanced protective mechanism in central nervous system development, pathological conditions and aging.

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation.

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity.

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

Small heat shock proteins determine synapse number and neuronal activity during development.

Environmental changes cause stress, Reactive Oxygen Species and unfolded protein accumulation which hamper synaptic activity and trigger cell death. Heat shock proteins (HSPs) assist protein refolding to maintain proteostasis and cellular integrity. Mechanisms regulating the activity of HSPs include transcription factors and posttranslational modifications that ensure a rapid response. HSPs preserve synaptic function in the nervous system upon environmental insults or pathological factors and contribute to the coupling between environmental cues and neuron control of development. We have performed a biased screening in Drosophila melanogaster searching for synaptogenic modulators among HSPs during development. We explore the role of two small-HSPs (sHSPs), sHSP23 and sHSP26 in synaptogenesis and neuronal activity. Both sHSPs immunoprecipitate together and the equilibrium between both chaperones is required for neuronal development and activity. The molecular mechanism controlling HSP23 and HSP26 accumulation in neurons relies on a novel gene (CG1561), which we name Pinkman (pkm). We propose that sHSPs and Pkm are targets to modulate the impact of stress in neurons and to prevent synapse loss.

Treading water: mixed effects of high fat diet on mouse behavior in the forced swim test.

Diet is an environmental factor with significant potential to affect the brain and behavior in both positive and negative ways. Work in animals is necessary to understand this relationship and how it may apply to mental health in humans. One area which has been investigated extensively is whether diet, specifically a high fat diet (HFD), can alter behavior in tasks, such as the forced swim test (FST) that assess stress coping. Therefore, we sought to analyze the literature regarding the effect of HFD on performance in the FST to determine whether there was a consistent effect of HFD on stress coping behavior. We conducted a Google Scholar search of English-language articles with the following terms: high fat diet, obesity, forced swim test, depression like behavior, mouse. Thirty studies from twenty-five publications are included in this survey. Fifteen studies were found where HFD had no effect on FST, 4 where HFD decreased immobility, and 11 where HFD increased immobility. Experimental details in these studies varied widely, including differences in the diet, mice, and experimental design. Additionally, we analyzed thirteen studies that performed the tail-suspension test (TST) after HFD, with six studies finding no change due to HFD and 7 reporting that HFD increased immobility. Further, 6 of these studies used both FST and TST with largely similar results in the two tasks, indicating concordance between the two tests of stress-coping behavior. We conclude that due to widely varying experimental details across studies no consistent effect of high fat diet on stress coping behavior can be determined at this point.

Adolescent microglia play a role in executive function in male mice exposed to perinatal high fat diet.

In humans, excessive gestational weight gain during pregnancy is associated with an increased risk for executive function deficits in the offspring. Our previous work has confirmed this finding in mice, as offspring from dams fed a 60% high fat (HF) diet during breeding, gestation, and lactation demonstrate impulsive-like behavior in the 5 choice serial reaction time task (5CSRTT). Because the prefrontal cortex (PFC), which plays a key role in executive function, undergoes substantial postnatal adolescent pruning and microglia are actively involved in synaptic refinement, we hypothesized that microglia may play a role in mediating changes in brain development after maternal HF diet, with a specific focus on microglial activity during adolescence. Therefore, we treated male and female offspring from HF or control diet (CD) dams with PLX3397-formulated diet (PLX) to ablate microglia during postnatal days 23-45. After PLX removal and microglial repopulation, adult mice underwent testing to evaluate executive function. Adolescent PLX treatment did increase the control male dropout rate in learning the basic FR1 task, but otherwise had a minimal effect on behavior in control offspring. In males, HF offspring learned faster and performed better on a simple operant task (fixed ratio 1) without an effect of PLX. However, in HF offspring this increase in FR1 responding was associated with more impulsive errors in the 5CSRTT while PLX eliminated this association and decreased impulsive errors specifically in HF offspring. This suggests that adolescent PLX treatment improves executive function and particularly impulsive behavior in adult male HF offspring, without an overall effect of perinatal diet. In females, maternal HF diet impaired reversal learning but PLX had no effect on performance. We then measured gene expression in adult male PFC, nucleus accumbens (NAC), and amygdala (AMG), examining targets related to synaptic function, reward, and inflammation. Maternal HF diet increased PFC synaptophysin and AMG psd95 expression. PFC synaptophysin expression was correlated with more impulsive errors in the 5CSRTT in the HF offspring only and PLX treatment eliminated this correlation. These data suggest that adolescent microglia may play a critical role in mediating executive function after perinatal high fat diet in males.

Correction: Let's call the whole thing off: evaluating gender and sex differences in executive function.

An error was made in our referencing.

Exposure to in utero inflammation increases locomotor activity, alters cognitive performance and drives vulnerability to cognitive performance deficits after acute immune activation.

Fetal exposure to intrauterine inflammation (IUI) affects brain development. Using intrauterine lipopolysaccharide (LPS) administration to induce a localized, rather than a systemic, inflammation, we have previously shown that IUI increases cytokine expression and microglia number, and reduces white matter in the brains of exposed offspring. Clinical data suggest that IUI may increase the risk for cognitive and neurodevelopmental disorders, however, IUI is often found in the context of preterm birth, making it difficult to disentangle the adverse effects of inflammation from those related to prematurity. Therefore, using a mouse model of IUI that does not involve preterm birth, operant tasks were used to evaluate motivation, attention, impulsivity, and locomotion. IUI-exposed offspring were found to have increased locomotion and increased motivation (females only), and testing in the 5-choice serial reaction time task (5-CSRTT) showed that IUI-exposed offspring performed more trials and could respond accurately at a shorter stimulus length. We have previously shown that IUI animals have a potentiated cytokine response to a "second hit" (acute LPS injection) in adulthood, so animals' performance in the 5CSRTT was evaluated following an acute injection of LPS. As opposed to the improved performance observed under baseline conditions, IUI exposed animals demonstrated a greater decrease in performance after an acute LPS administration. To identify putative molecular mechanisms underlying this potentiated decline in cognitive performance, PFC samples were collected immediately after post-LPS cognitive testing and targeted gene expression analysis was correlated with specific measures of cognitive performance. Three receptors important for neuron-microglia crosstalk were found to correlate with task performance in the males following acute LPS administration. These data demonstrate that early life exposure to localized inflammation of the uterus, in the absence of prematurity, increases locomotor activity and improves some aspects of cognitive performance, but drives a vulnerability for adult cognitive performance deficits in response to acute infection.