A TNF-α inhibitor abolishes sepsis-induced cognitive impairment in mice by modulating acetylcholine and nitric oxide homeostasis, BDNF release, and neuroinflammation.

Neurodegenerative disorders have a pathophysiology that heavily involves neuroinflammation. In this study, we used lipopolysaccharide (LPS) to create a model of cognitive impairment by inducing systemic and neuroinflammation in experimental animals. LPS was injected intraperitoneally at a dose of 0.5 mg/kg during the last seven days of the study. Adalimumab (ADA), a TNF-α inhibitor, was injected at a dose of 10 mg/kg a total of 3 times throughout the study. On the last two days of the experiment, 50 mg/kg of curcumin was administered orally as a positive control group. Open field (OF) and elevated plus maze tests (EPM) were used to measure anxiety-like behaviors. The tail suspension test (TST) was used to measure depression-like behaviors, while the novel object recognition test (NOR) was used to measure learning and memory activities. Blood and hippocampal TNF α and nitric oxide (NO) levels, hippocampal BDNF, CREB, and ACh levels, and AChE activity were measured by ELISA. LPS increased anxiety and depression-like behaviors while decreasing the activity of the learning-memory system. LPS exerted this effect by causing systemic and neuroinflammation, cholinergic dysfunction, and impaired BDNF release. ADA controlled LPS-induced behavioral changes and improved biochemical markers. ADA prevented cognitive impairment induced by LPS by inhibiting inflammation and regulating the release of BDNF and the cholinergic pathway.

N-acetylcysteine ameliorates chemotherapy-induced impaired anxiety and depression-like behaviors by regulating inflammation, oxidative and cholinergic status, and BDNF release.

Mood disorders caused by chemotherapy have become more important as the survival of cancer patients increases, and new studies in this field will contribute to the prevention of this disorder. For this purpose, we used methotrexate, a chemotherapeutic agent frequently preferred in oncological cases. Mtx was administered as a single dose of 100 mg/kg intraperitoneally to male Wistar albino rats. Since oxidative stress plays an important role in chemotherapy-induced emotional impairment, n-acetylcysteine (NAC), a potent antioxidant, was administered at 500 mg/kg in two doses before Mtx administration. We evaluated anxiety and depression-like behaviors 24 h after Mtx administration, as well as some oxidative and inflammatory markers in blood serum and hippocampal tissue, acetylcholinesterase activity (AChE), and brain-derived neurotrophic factor (BDNF) release in hippocampal tissue. In rats, Mtx induced anxiety and depression-like behaviors as well as abnormalities in oxidative and inflammatory markers in blood serum and hippocampal tissue, increased AChE activity in hippocampal tissue, and decreased BDNF release. NAC treatment was found to ameliorate Mtx-induced anxiety and depression-like behaviors, increase antioxidant capacity, reduce oxidative stress and inflammatory response, and regulate AChE activity and BDNF release. In conclusion, the fact that NAC treatment of Mtx was effective is important for revising the treatment strategies for individuals suffering from this disorder, and this effect is thought to be related to the antioxidant and anti-inflammatory power of NAC.

Carvacrol Reduces Mercuric Chloride-Induced Testicular Toxicity by Regulating Oxidative Stress, Inflammation, Apoptosis, Autophagy, and Histopathological Changes.

Mercuric chloride (HgCl2) is a heavy metal that is toxic to the human body. Carvacrol (CAR) is a flavonoid found naturally in plants and has many biological and pharmacological activities including anti-inflammatory, antioxidant, and anticancer activities. This study aimed to investigate the efficacy of CAR in HgCl2-induced testicular tissue damage. HgCl2 was administered intraperitoneally at a dose of 1.23 mg/kg body weight alone or in combination with orally administered CAR (25 mg/kg and 50 mg/kg body weight) for 7 days. Biochemical and histological methods were used to investigate oxidative stress, inflammation, apoptosis, and autophagy pathways in testicular tissue. CAR treatment increased HgCl2-induced decreased antioxidant enzyme (SOD, CAT, and GPx) activities and GSH levels. In addition, CAR reduced MDA levels, a marker of lipid peroxidation. CAR decreased the levels of inflammatory mediators NF-κB, TNF-α, IL-1ß, COX-2, iNOS, MAPK14, MAPK15, and JNK. The increases in apoptotic Bax and Caspase-3 with HgCl2 exposure decreased with CAR, while the decreased antiapoptotic Bcl-2 level increased. CAR reduced HgCl2-induced autophagy damage by increasing Beclin-1, LC3A, and LC3B levels. Overall, the data from this study suggested that testicular tissue damage associated with HgCl2 toxicity can be mitigated by CAR administration.

An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

The protective effects of chrysin on cadmium-induced pulmonary toxicity; a multi-biomarker approach.

This study aimed to determine the potential protective effects of chrysin (CHR) on experimental cadmium (Cd)-induced lung toxicity in rats. To this end, rats were divided into five groups; Control, CHR, Cd, Cd + CHR25, Cd + CHR50. In the study, rats were treated with CHR (oral gavage, 25 mg/kg and 50 mg/kg) 30 min after giving Cd (oral gavage, 25 mg/kg) for 7 consecutive days. The effects of Cd and CHR treatments on oxidative stress, inflammatory response, ER stress, apoptosis and tissue damage in rat lung tissues were determined by biochemical and histological methods. Our results revealed that CHR therapy for Cd-administered rats could significantly reduce MDA levels in lung tissue while significantly increasing the activity of antioxidant enzymes (SOD, CAT, GPx) and GSH levels. CHR agent exerted antiinflammatory effect by lowering elevated levels of NF-κB, IL-1ß IL-6, TNF-α, RAGE and NRLP3 in Cd-induced lung tissue. Moreover CHR down-regulated Cd-induced ER stress markers (PERK, IRE1, ATF6, CHOP, and GRP78) and apoptosis markers (Caspase-3, Bax) lung tissue. CHR up-regulated the Bcl-2 gene, an anti-apoptotic marker. Besides, CHR attenuated the side effects caused by Cd by modulating histopathological changes such as hemorrhage, inflammatory cell infiltration, thickening of the alveolar wall and collagen increase. Immunohistochemically, NF-κB and Caspase-3 expressions were intense in the Cd group, while these expressions were decreased in the Cd + CHR groups. These results suggest that CHR exhibits protective effects against Cd-induced lung toxicity in rats by ameliorating oxidative stress, inflammation, apoptosis, endoplasmic reticulum stress and histological changes.

CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance.

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 - 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

The effect of isotretinoin therapy on oxidative damage in rats.

Isotretinoin is prescribed in many dermatologic disorders, but mostly in acne. There is limited research about oxidative stress induced by isotretinoin and its effects on the liver tissue, muscle tissue, and blood. In this study, oxidative damage of isotretinoin on the liver, muscles, and blood in rats at the therapeutic dosage for humans, is evaluated. Thirty, 2-months-old Wistar albino rats were randomly divided into four groups. Isotretinoin was administered at the human equivalent low dose of 7.5 mg/kg by gavage. Blood, liver, and skeletal muscle samples were taken from the animals under anesthesia. Oxidative stress and antioxidant defense markers such as Malondialdehyde (MDA), Protein carbonyl (PC), 8-OHDG (8-hydroxy-deoxyguanosine), SOD (Superoxide dismutase), GSH(Glutathione), GPX (glutathione peroxidase), NO (Nitric Oxide) levels, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and creatine kinase (CK) levels were measured. There were significant differences between the ALT values of the control group and the third month of isotretinoin treatment group. Oxidative stress markers such as 8-OHDG, PC, GSH, GPX, and NO values significantly differed in month 3. SOD was significantly lower in the treatment groups compared to the control group. Our study supports that the levels of oxidative markers are increasing with the isotretinoin treatment so this may flare acne. GPX levels increased at the muscle tissue level, and may be responsible for the myopathy that is seen in acne patients. Addition of antioxidants to isotretinoin treatment may be beneficial in reducing oxidative damage.

Eastern Anatolian apples with a unique population structure are genetically different from Anatolian apples.

The origin of the apple is known to be the Transcaucasian region. Eastern Anatolia, which is located on the migration routes from Asia to Europe, has a rich and an uncharacterized apple germplasm and the characterization of apple genetic sources from this region is important for both evolutionary studies and apple breeding. In this study, 94 M. domestica accessions originated from seven diverse regions within Eastern Anatolia were studied using 16 SSR (simple sequence repeat) loci. SSR markers we used produced high allele numbers in all loci and CH02d11 (PI: 0.059) with 18 alleles was the most informative locus. In addition, 14 identical accession groups were identified. Most likely due to self-incompatibility, relatively high levels of heterozygosity (Ho: 0.696) was found for Eastern Anatolia apples. Structure Harvester analyses of East Anatolian apple accessions showed that although each group seems to be somewhat distinct, some levels of admixture with other populations might also exist. Due to a significant gene flow between all pairs of seven apple populations, a limited (low) differentiation was found between the populations. Comparisons using 16 common SSR loci revealed that Eastern Anatolia accessions were genetically different from Anatolian accessions. In addition, based on FCA, and Nei's genetic distance analyses, Eastern Anatolian apples were found to be genetically different from the commercial apple cultivars Golden Delicious and Florina. Our results suggesting that Eastern Anatolia apple populations have a unique structure will be useful for future genetic and evolutionary studies on apples.

Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37.

The peptide LL-37, a component of the human innate immune system, represents a promising drug candidate. In particular, the development of low-cost production platform technology is a critical bottleneck in its use in medicine. In the present study, a viable approach for the LL-37 production in transgenic barley is developed. First, comparative analyses of the effects of different fused peptide epitope tags applicable for accumulation and purification on LL-37 production yield are performed using transient expression in tobacco leaves. Following the selection of the most yielding fusion peptide strategies, eight different constructs for the expression of codon optimized chimeric LL-37 genes in transgenic barley plants are created. The expression of individual constructs is driven either by an endosperm-specific promoter of the barley B1 hordein gene or by the maize ubiquitin promoter. The transgenes are stably integrated into the barley genome and inherited in the subsequent generation. All transgenic lines show normal phenotypes and are fertile. LL-37 accumulated in the barley seeds up to 0.55 mg per 1 kg of grain. The fused epitope tags are cleaved off by the use of enterokinase. Furthermore, in planta produced LL-37 including the fused versions is biologically active.

3',4'-Dihydroxyflavonol attenuates spatial learning and memory impairments in global cerebral ischemia.

OBJECTIVES: In the present study, effects of 3',4'-dihydroxyflavonol (DiOHF) on anxiety-like behavior, and learning and memory were investigated in a model of transient global cerebral ischemia and reperfusion. METHODS: The animals were assigned to sham-operated, ischemia, and two DiOHF-treated (10 mg/kg i.p.) groups. DiOHF was administered at 1 hour before and immediately after the ischemia. Male rats were subjected to bilateral common carotid artery occlusion to induce acute cerebral ischemia for 20 minutes, followed by reperfusion for 7 days. The openfield, elevated plus maze (EPM), and Morris water maze tests were used to evaluate the effects of DiOHF treatment on ischemia-induced locomotor activity, anxiety-like behavior, and spatial and recognition memory impairments, respectively. RESULTS: In the open field test, locomotor activity in the ischemic rats was not altered 6 days after the ischemia, nor was anxiety-like behavior, which was evaluated with the EPM (P > 0.05). In the water-maze test, cerebral ischemia significantly decreased the exploration time in the target quadrant, and the platform crossing counts were lower (P < 0.05) in the probe trial test; this memory impairment was significantly improved by DiOHF applied 1 hour before and immediately after ischemia (P < 0.05). DISCUSSION: All together, these findings suggest that DiOHF reverses spatial learning and memory deficits resulting from transient global ischemia but has no significant effect on anxiety-like behavior.

Impact of enriched environment on production of tau, amyloid precursor protein and, amyloid-ß peptide in high-fat and high-sucrose-fed rats.

OBJECTIVE: The Western-type diet is associated with an elevated risk of Alzheimer's disease and other milder forms of cognitive impairment. The aim of the present study was to investigate the effects of the environmental enrichment on amyloid and tau pathology in high-fat and high-sucrose-fed rats. METHODS: In total, 40 adult male rats were categorised into two main groups according to their housing conditions: enriched environment (EE, n=16) and standard housing condition (n=24). The groups were further divided into five subgroups that received standard diet, high-fat diet, and high-sucrose diet. We performed the analysis of amyloid ß-peptide (Aß) (1-40), Aß(1-42), amyloid precursor protein (APP), and tau levels in the hippocampus of rats that were maintained under standard housing conditions or exposed to an EE. RESULTS: The EE decreased the Aß(1-40), Aß(1-42), APP, and tau levels in high-fat and high-sucrose-fed rats. CONCLUSION: This observation shows that EE may rescue diet-induced amyloid and tau pathology.

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria.

Environmental enrichment reverses cognitive impairments provoked by Western diet in rats: Role of corticosteroid receptors.

AIMS: Previous studies demonstrated that the Western diet (WD), which is rich in saturated fat (HFD) and refined sugar (HSU), is related to the impairments of hippocampus-dependent learning and memory and forebrain synaptic plasticity in rodents. The environmental enrichment (EE) has been shown to enhance learning and memory in the HFD-induced cognitive deficits, but the exact mechanism is still not clearly understood. Therefore, the present study aimed to clarify the effects of the EE on spatial memory in WD-fed rats, and to analyze the potential role of corticosteroid receptors in the EE conditioning. MAIN METHODS: Male Wistar albino rats were housed in either an enriched or standard environment and fed with the HFD (35% of energy as fat), HSU (100% of carbohydrate as sucrose) or standard rat chow for 4weeks. We used the Morris' water maze test (MWM) to assess the learning and memory performance, and measured plasma levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH), as well as glucocorticoid (GR) and mineralocorticoid receptor (MR) levels in the hippocampus. KEY FINDINGS: The results showed that HFD-fed rats displayed poorer learning and memory performance evaluated with MWM than controls. The EE reversed the cognitive deficits caused by the HFD. In addition, the EE resulted in an increase of GR and MR levels without affecting plasma CORT and ACTH concentrations. SIGNIFICANCE: Based on these findings, it could be suggested that the EE plays an important role in amelioration of the HFD-induced cognitive impairments, but this intervention is independent of the hypothalamic-pituitary-adrenal axis and hippocampal corticosteroid receptor levels.

Antidepressant-like effects of quercetin in diabetic rats are independent of hypothalamic-pituitary-adrenal axis.

OBJECTIVE: Quercetin, one of the most potent flavonol in the family of flavonoids, has been shown to have benefits against diabetes and its complications. In the present study, we investigated effects of quercetin on depression-like behaviours and hypothalamic-pituitary-adrenal (HPA) axis in diabetic rats. METHODS: Experimental diabetes was induced by using streptozotocin, and either 50 or 100 mg/kg quercetin was intraperitoneally administered for 21 days. Following the last treatment, animals were subjected to the forced swim test, and subsequently, the blood was obtained by cardiac puncture to measure plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels. RESULTS: A significant increase of the total immobile time, accompanied by a decrease in the immobility latency, which suggests a depressive status, was observed in diabetic animals that was reversed by the treatment of 50 mg/kg quercetin. However, the higher dose of quercetin (100 mg/kg) was ineffective in alleviating depression-like behaviours. The plasma concentrations of ACTH, and total- and free-CORT were not affected by both doses of quercetin. CONCLUSION: Therefore, we concluded that the antidepressant-like effects of quercetin in diabetes are independent of the HPA axis.

The Effects of Electrocautery on Peripheral Nerve: An Experimental Study.

BACKGROUND The aim of this study was to assess the usability of an electrocautery device as nerve stimulator and to investigate histopathologically the adverse effects of electrocautery at low power on rat sciatic nerves. METHODS A total of 36 female Sprague-Dawley albino rats were divided into six groups according to the power applied to their sciatic nerves (1, 2, 3, 4, 5 and 6 W, respectively). Pathologic changes were studied by microscopic examination and scored (no change = 0, mild = 1, moderate = 2, severe = 3). Multiple comparisons were provided for all groups by the Bonferroni test (one-way analysis of variance). A p value < 0.05 was accepted as statistically significant. RESULTS The average scores were 2.66 ± 0.51, 3.66 ± 0.51, 5.83 ± 1.83, 10.0 ± 1.78, 11.0 ± 1.54, and 13.8 ± 0.89 in groups 1 to 6, respectively. Significant differences were found between all groups (p < 0.01), except between groups 1 and 2, groups 2 and 3, and groups 4 and 5 (p > 0.05) Variable motor responses and foot deformities were observed at the different power levels. CONCLUSION Although electrocautery devices provoke motor responses if getting in contact with peripheral nerves as do nerve stimulators, their use induces histopathologically adverse effects even at the lowest power. Their use around peripheral nerves should be avoided.

Diversity in boron toxicity tolerance of Australian barley (Hordeum vulgare L.) genotypes.

BACKGROUND: Boron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1. RESULTS: We investigated sequence and expression level diversity in HvBot1 and HvNIP2;1 across barley germplasm, and identified five novel coding sequence alleles for HvBot1. Lines were identified containing either single or multiple copies of the Sahara HvBot1 allele. We established that only the tandemly duplicated Sahara allele conferred B tolerance, and this duplicated allele was found only in a set of nine lines accessioned in Australian collections as Sahara 3763-3771. HvNIP2;1 coding sequences were highly conserved across barley germplasm. We identified the likely causative SNP in the 5'UTR of Sahara HvNIP2;1, and propose that the creation of a small upstream open reading frame interferes with HvNIP2;1 translation in Sahara 3771. Similar to HvBot1, the tolerant HvNIP2;1 allele was unique to the Sahara barley accessions. We identified a new source of the 2H B tolerance allele controlling leaf symptom development, in the landrace Ethiopia 756. CONCLUSIONS: Ethiopia 756, as well as the cultivar Sloop Vic which carries both the 2H and HvBot1 B tolerance alleles derived from Sahara 3771, may be valuable as alternative parents in breeding programs targeted to high soil B environments. There is significant diversity in B toxicity tolerance among contemporary Australian barley varieties but this is not related to variation at any of the four known B tolerance loci, indicating that novel, as yet undiscovered, sources of tolerance exist.

Curcumin alleviates cisplatin-induced learning and memory impairments.

The present study has been designed to investigate the role of curcumin on cisplatin-inducedcognitive impairment and to reveal mechanisms of cisplatin's detrimental actions on cognition in rats. Animals were treated with cisplatin (5mg/kg/week) and/or curcumin (300mg/kg/day) for 5weeks. Morris water maze test was used to assess spatial learning and memory. Enzymatic activities of acetylcholinesterase (AChE) and superoxide dismutase (SOD) were evaluated from hippocampus and plasma samples, and malondialdehyde (MDA), which is the end-product of lipid peroxidation, was determined by a colorimetric method. Our results showed that cisplatin (5mg/kg/week, 5weeks) caused learning and memory deficits, elevated MDA content, decreased SOD activity in the hippocampus and plasma, and AChE activity in the hippocampus. Curcumin improved learning and memory in rats with administration of cisplatin. In addition, curcumin significantly reduced the level of MDA and increased the activities of SOD and AChE. Taken together, our findings indicate that curcumin ameliorates cisplatin-induced spatial learning and memory impairment, possibly through restored cholinergic function and enhanced oxidative status.

Curcumin improves spatial memory and decreases oxidative damage in aged female rats.

Aging induced cognitive impairment has been well documented for many years and several antioxidant strategies have been developed against this impairment. Curcumin is the active component of curcuma longa and has shown antioxidant, antiinflamatory and neuroprotective properties. We hypothesized that curcumin would have an influence on cognitive functions in aged female rats. The purpose of the present study was to investigate the effects of curcumin supplementation on cognitive impairment evaluated by Morris water maze (MWM) as well as the oxidative stress induced by aging in female rats. Rats were randomly divided into either control or curcumin-supplemented groups. Curcumin or vehicle (corn oil) were given once daily for a period of 12 days, beginning 7 days prior to and 5 days during the behavioral tests. Behavioral assessment was performed in MWM. At the end of the behavioral test, blood samples and brain tissues were taken for the analysis of malondialdeyde (MDA), protein carbonyl and glutathione levels. During the training session, curcumin supplementation decreased latency to reach to the platform and the total distance traveled. During the probe trial, curcumin supplementation increased the number of platform crossings. In addition to the behavioral testing, biochemical results showed that MDA levels decreased in brain tissue by curcumin supplementation. It may be concluded that, curcumin supplementation improves cognitive functions by decreasing the lipid peroxidation in brain tissue of aged female rats.