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A histidine acid phosphatase (HAP) (PhySc) with 99.50% protein sequence similarity with PHO5 from Saccharomyces cerevisiae was expressed functionally with the molecular mass of ∼110 kDa through co-expression along with the set of molecular chaperones dnaK, dnaJ, GroESL. The purified HAP illustrated the optimum activity of 28.75 ± 0.39 U/mg at pH 5.5 and 40 ˚C. The Km and Kcat values towards calcium phytate were 0.608 ± 0.09 mM and 650.89 ± 3.6 s- 1. The half-lives (T1/2) at 55 and 60 ˚C were 2.75 min and 55 s, respectively. The circular dichroism (CD) demonstrated that PhySc includes 30.5, 28.1, 21.3, and 20.1% of random coils, α-Helix, ß-Turns, and ß-Sheet, respectively. The Tm recorded by CD for PhySc was 56.5 ± 0.34˚C. The molecular docking illustrated that His59 and Asp322 act as catalytic residues in the PhySc. MD simulation showed that PhySc at 40 ˚C has higher structural stability over those of the temperatures 60 and 80 ˚C that support the thermodynamic in vitro investigations. Secondary structure content results obtained from MD simulation indicated that PhySc consists of 34.03, 33.09, 17.5, 12.31, and 3.05% of coil, helix, turn, sheet, and helix310, respectively, which is almost consistent with the experimental results.

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The oriental tobacco budworm Helicoverpa assulta (Lepidoptera: Noctuidae) is a specialist pest that may cause serious damages to important crops such as chili pepper and tobacco. Various man-made insecticides have been applied to control the infestation of this pest. To understand how this pest copes with insecticides, it is required to identify key players involved in insecticide transformation. In this study, a P450 gene of CYP6B subfamily was identified in the oriental tobacco budworm, and its expression pattern was revealed. Moreover, the activities of HassCYP6B6 against 12 insecticides were explored using recombinant enzymes produced in the facile Escherichia coli. Data from metabolic experiments showed that HassCYP6B6 was able to metabolize conventional insecticides including organophosporates (diazinon, malathion, phoxim), carbamate propoxur, and pyrethroid esfenvalerate, while no significant metabolism was observed towards new-type pesticides such as neonicotinoids (acetamiprid, imidacloprid), diamides (chlorantraniliprole, cyantraniliprole), macrocyclic lactone (emamectin benzoate, ivermectin), and metaflumizone. Structures of metabolites were proposed based on mass spectrometry analyses. The results demonstrate that HassCYP6B6 plays important roles in the transformation of multiple insecticides via substrate-dependent catalytic mechanisms including dehydrogenation, hydroxylation and oxidative desulfurization. The findings have important applied implications for the usage of insecticides.

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Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

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We investigated the molecular and biochemical properties of two acetylcholinesterases (FoAChE1 and FoAChE2) from the Western flower thrips, Frankliniella occidentalis. Polyacrylamide gel electrophoresis and western blotting confirmed the membrane-anchored nature of both FoAChE1 and FoAChE2, which was further supported by hydrophobicity and glycophosphatidylinositol anchor predictions. High expression levels of both enzymes were observed in the head, indicating their predominant distribution in neuronal tissues. FoAChE1 exhibited significantly higher expression levels in all examined tissues compared to FoAChE2, suggesting its major role as a synaptic enzyme. Nonetheless, both recombinant enzymes displayed robust catalytic activity toward acetylthiocholine iodide, and FoAChE1 demonstrated nearly identical catalytic efficiency compared to FoAChE2. FoAChE1 exhibited slightly lower sensitivities to the cholinesterase inhibitors tested, including organophosphates (OPs) and carbamates (CBs), compared to FoAChE2. Field populations of F. occidentalis exhibited polymorphism of alanine vs. serine at position 197 of FoAChE1 within the conserved oxyanion hole. Contrary to common belief, however, functional analysis using recombinant enzymes revealed that neither A197 nor S197 residue was associated with FoAChE1 insensitivity to OPs and CBs. FoAChE2 did not exhibit any polymorphic amino acid substitutions at the positions known to be associated with resistance. Due to the absence of apparent resistance-associated mutations in field populations of F. occidentalis, the judicious use of some OPs or CBs can be suggested for controlling the highly resistant populations to other insecticides. Overall, our findings highlight the significance of both FoAChE1 and FoAChE2 as targets for toxicity assessment, while the specific contribution of each enzyme to toxicity remains unclear.

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Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.

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The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In the current study, two isoforms of a novel ammonia transporter, LpHIAT1α and LpHIAT1ß, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other, and 77-86% amino acid homology to HIAT1 found in fish and crustaceans. In L. polyphemus, both isoforms were expressed in the gills, coxal glands, and brain. Slightly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and brain compared to the LpHIAT1ß isoform. A functional expression analysis of LpHIAT1α and LpHIAT1ß in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine when compared to controls, indicating an ammonia excretory function of the proteins. Exposure to elevated environmental ammonia (HEA, 1 mmol l-1 NH4Cl) caused an increase in mRNA expression of LpHIAT1ß in the ion-conductive ventral gill half. High mRNA expression of both isoforms in the brain, and the observation that LpHIAT1α and LpHIAT1ß likely mediate cellular ammonia excretion, suggests that these highly conserved ammonia transporters have an important housekeeping function in cellular ammonia elimination.

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Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short-chain prenyltransferases catalyse the formation of the acyclic precursors (E)-FPP and (E)-GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate-rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC-MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC-MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)-FPP using DMAPP or (E)-GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)-GPP or (E)-FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid.

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Post-translational modifications (PTMs) are protein modifications that occur after protein biosynthesis, playing a crucial role in regulating protein function. They are involved in the functional expression of G-protein-coupled receptors (GPCRs), as well as intracellular and secretory protein signaling. Here, we aimed to investigate the PTMs of the apelin receptor (APLNR), a GPCR and their potential influence on the receptor's function. In an in vitro experiment using HEK cells, we only observed glycosylation as a PTM of the APLNR and ineffective receptor signaling by the agonist, (Pyr1)-apelin-13. In contrast, when analyzing mouse spinal cord, we detected glycosylation and other PTMs, excluding isopeptidation. This suggests that additional PTMs are involved in the functional expression of the APLNR in vitro. In summary, these findings suggest that the APLNR in vivo requires multiple PTMs for functional expression. To comprehensively understand the pharmacological effects of the APLNR, it is essential to establish an in vitro system that adequately replicates the receptor's PTM profile. Nonetheless, it is crucial to overcome the challenge of heat-sensitive proteolysis in APLNR studies. By elucidating the regulation of PTMs, further research has the potential to advance the analysis and pharmacological studies of both the apelin/APLNR system and GPCR signal modulation.

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A new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant known as Omicron has caused a rapid increase in recent global patients with coronavirus infectious disease 2019 (COVID-19). To overcome the COVID-19 Omicron variant, production of a recombinant spike receptor binding domain (RBD) is vital for developing a subunit vaccine or a neutralizing antibody. Although bacterial expression has many advantages in the production of recombinant proteins, the spike RBD expressed in a bacterial system experiences a folding problem related to disulfide bond formation. In this study, the soluble Omicron RBD was obtained by a disulfide isomerase-assisted periplasmic expression system in Escherichia coli. The Omicron RBD purified from E. coli was very well recognized by anti-SARS-CoV-2 antibodies, sotrovimab (S309), and CR3022, which were previously reported to bind to various SARS-CoV-2 variants. In addition, the kinetic parameters of the purified Omicron RBD upon binding to the human angiotensin-converting enzyme 2 (ACE2) were similar to those of the Omicron RBD produced in the mammalian expression system. These results suggest that an E. coli expression system would be suitable to produce functional and correctly folded spike RBDs of the next emerging SARS-CoV-2 variants quickly and inexpensively.

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The antitumor drug doxorubicin is widely used in clinical practice. However, the low yield and high cost of this drug highlight the urgent need for cost-effective processes to rapidly manufacture antitumor drugs at scale. In the biosynthesis pathway, the multi-functional cytochrome P450 enzyme DoxA catalyzes the last three steps of hydroxylation. The final conversion of daunorubicin to doxorubicin is the rate-limiting step. In our work, the DoxA has been expressed with the ferredoxin reductase FDR2 and the ferredoxin FDX1 and purified to homogeneous. The reduced carbon monoxide difference spectroscopy, heme concentration, and enzymatic characteristic were characterized. These studies suggest an approach for engineering Streptomyces P450s with functional expression for mechanistic and structural studies.

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Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the α5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the α5 receptor has low sensitivity to acetylcholine with an EC50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC50 at 119 µM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the α5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides.

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BACKGROUND: This study aimed to analyze the in vitro effect of micro-strain stress on the proliferation and functional marker expression in chondrocytes isolated from human osteoarthritis cartilage samples. METHODS: Chondrocytes isolated from human osteoarthritis cartilage samples were subjected to loading with different types of micro-strain stress. The proliferation activity was assessed by flow cytometry, and the functional expression of chondrocyte markers was detected by qRT-PCR and western blot. RESULTS: Flow cytometry results showed stimulation of proliferation of human osteoarthritic chondrocytes when an adequate micro-strain stress was applied. qRT-PCR and western blot results showed that micro-strain stress promotes human osteoarthritic chondrocyte functional marker expression. These features coincide with the upregulation of multiple proteins and genes affecting cell proliferation and functional chondrocyte marker expression, including cyclin D1, collagen II, and Rock. CONCLUSION: Adequate micro-strain stress could activate the Rho/Rock signaling pathway in osteoarthritic chondrocytes, thus transmitting mechanical signals to the cytoskeleton. This process leads to cytoskeleton reorganization, and transmission of the mechanical signals to the downstream effectors to promote proliferation and functional marker expression of osteoarthritic chondrocytes.

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Insect nicotinic acetylcholine receptors (nAChR) are molecular targets of highly effective insecticides. The use of chaperone proteins has been key to successful functional expression of these receptors in heterologous systems, permitting functional and pharmacological studies of insect nAChRs with particular subunit composition. Here, we report the first use of the chaperone protein, NACHO, to enable functional expression of an insect nAChR, the α6 subunit from Apis mellifera, in Xenopus laevis oocytes. This is also the first report of functional expression of a homomeric insect α6 nAChR. Using two-electrode voltage-clamp electrophysiology we show that the acetylcholine EC50 of the α6 receptor is 0.88 µM and that acetylcholine responses are antagonized by α-bungarotoxin. Spinosad showed agonist actions and kept the ion channel open when co-applied with acetylcholine, reinforcing the α6 nAChR subunit as a key molecular target for the spinosyn class of insecticide. The use of NACHO may provide a basis for future expression studies of insect α6 nAChRs, potentially providing a tool for the discovery of novel insecticides.

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Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

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BACKGROUND: One goal of expression data analysis is to discover the biological significance or function of genes that are differentially expressed. Gene Set Enrichment (GSE) analysis is one of the main tools for function mining that has been widely used. However, every gene expressed in a cell is valuable information for GSE for single-cell RNA sequencing (scRNA-SEQ) data and not should be discarded. METHODS: We developed the functional expression matrix (FEM) algorithm to utilize the information from all expressed genes. The algorithm converts the gene expression matrix (GEM) into a FEM. The FEM algorithm can provide insight on the biological significance of a single cell. It can also integrate with GEM for downstream analysis. RESULTS: We found that FEM performed well with cell clustering and cell-type specific function annotation in three datasets (peripheral blood mononuclear cells, human liver, and human pancreas).

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Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

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The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide ('GRKKRRQRRR') can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B-I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B-I and promoted LC3B-I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.

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A high serum uric acid (SUA) concentration is associated with hyperuricemia (HUA) and gout. In order to obtain long-acting therapeutic effect, correction of purine metabolism at genetic level is advantageous. For this purpose, we expressed three "human-like" urate oxidases in human hepatocytes (HL-7702) by lentivirus-mediated transduction. Enzymatic assay revealed that the recombinant urate oxidases expressed in HL-7702 cells were functionally active. Electron microscopy study showed that the recombinant enzymes were localized to peroxisome and formed distinct crystalloid core structures as in other mammal cells. Although similar rate of uric acid degradation was observed for all recombinant urate oxidases, HL-7702-pLVX-UOX83 cells and HL-7702-pLVX-UOX214/217 cells retained more cell viability compared with HL-7702-pLVX-UOXPBC at high uric acid level. This study provides a new direction for the treatment of gout and hyperuricemia.

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Cadmium (Cd) is an extremely toxic environmental pollutant with high mobility in soils, which can contaminate groundwater, increasing its risk of entering the food chain. Yeast biosorption can be a low-cost and effective method for removing Cd from contaminated aqueous solutions. We transformed wild-type Saccharomyces cerevisiae (WT) with two versions of a Populus trichocarpa gene (PtMT2b) coding for a metallothionein: one with the original sequence (PtMT2b 'C') and the other with a mutated sequence, with an amino acid substitution (C3Y, named here: PtMT2b 'Y'). WT and both transformed yeasts were grown under Cd stress, in agar (0; 10; 20; 50 µM Cd) and liquid medium (0; 10; 20 µM Cd). Yeast growth was assessed visually and by spectrometry OD600. Cd removal from contaminated media and intracellular accumulation were also quantified. PtMT2b 'Y' was also inserted into mutant strains: fet3fet4, zrt1zrt2 and smf1, and grown under Fe-, Zn- and Mn-deficient media, respectively. Yeast strains had similar growth under 0 µM, but differed under 20 µM Cd, the order of tolerance was: WT < PtMT2b 'C' < PtMT2b 'Y', the latter presenting 37% higher growth than the strain with PtMT2b 'C'. It also extracted ~80% of the Cd in solution, and had higher intracellular Cd than WT. Mutant yeasts carrying PtMT2b 'Y' had slightly higher growth in Mn- and Fe-deficient media than their non-transgenic counterparts, suggesting the transgenic protein may chelate these metals. S. cerevisiae carrying the altered poplar gene offers potential for bioremediation of Cd from wastewaters or other contaminated liquids.

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This study was designed to demonstrate that prenatal ethanol exposure (PEE) can induce low functional expression of the hypothalamus in male offspring rats and explore the underlying mechanism. Pregnant rats were administered 4 g/kg ethanol or normal saline by oral gavage each day from gestational day (GD) 9 to GD20. Male GD20 foetuses and postnatal day 120 adult offspring rats were sacrificed under anaesthesia. Hypothalamic cells from male GD20~postnatal day (PD) 7 rats were treated with different doses of corticosterone and the glucocorticoid receptor (GR) antagonist mifepristone for 5 days. In this study, we found that PEE-induced overexposure of maternal glucocorticoids enhanced the expression of L-glutamic acid decarboxylase (GAD) 67 in the hypothalamic paraventricular nucleus (PVN) by activating the glucocorticoid metabolic activation system, further inducing the conversion of glutamate to L-gamma-aminobutyric acid (GABA) and developmental imbalance of glutamatergic/GABAergic projections to the PVN. The imbalance change was maintained until after birth, resulting in the inhibition of parvocellular neurons and low functional expression of the hypothalamus in PEE offspring rats. Our study indicated that low functional expression of the hypothalamus in male PEE offspring rats was associated with developmental programming of an imbalance of glutamatergic/GABAergic projections to the PVN.

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