Role of Polyinosinic:Polycytidylic Acid-Induced Maternal Immune Activation and Subsequent Immune Challenge in the Behaviour and Microglial Cell Trajectory in Adult Offspring: A Study of the Neurodevelopmental Model of Schizophrenia

Multiple lines of evidence support the pathogenic role of maternal immune activation (MIA) in the occurrence of the schizophrenia-like disturbances in offspring. While in the brain the homeostatic role of neuron-microglia protein systems is well documented, the participation of the CX3CL1-CX3CR1 and CD200-CD200R dyads in the adverse impact of MIA often goes under-recognized. Therefore, in the present study, we examined the effect of MIA induced by polyinosinic:polycytidylic acid (Poly I:C) on the CX3CL1-CX3CR1 and CD200-CD200R axes, microglial trajectory (MhcII, Cd40, iNos, Il-1β, Tnf-α, Il-6, Arg1, Igf-1, Tgf-β and Il-4), and schizophrenia-like behaviour in adult male offspring of Sprague-Dawley rats. Additionally, according to the “two-hit” hypothesis of schizophrenia, we evaluated the influence of acute challenge with Poly I:C in adult prenatally MIA-exposed animals on the above parameters. In the present study, MIA evoked by Poly I:C injection in the late period of gestation led to the appearance of schizophrenia-like disturbances in adult offspring. Our results revealed the deficits manifested as a diminished number of aggressive interactions, presence of depressive-like episodes, and increase of exploratory activity, as well as a dichotomy in the sensorimotor gating in the prepulse inhibition (PPI) test expressed as two behavioural phenotypes (MIA_PPI-low and MIA_PPI-high). Furthermore, in the offspring rats subjected to a prenatal challenge (i.e., MIA) we noticed the lack of modulation of behavioural changes after the additional acute immune stimulus (Poly I:C) in adulthood. The important finding reported in this article is that MIA affects the expression and levels of the neuron-microglia proteins in the frontal cortex and hippocampus of adult offspring. We found that the changes in the CX3CL1-CX3CR1 axis could affect microglial trajectory, including decreased hippocampal mRNA level of MhcII and elevated cortical expression of Igf-1 in the MIA_PPI-high animals and/or could cause the up-regulation of an inflammatory response (Il-6, Tnf-α, iNos) after the “second hit” in both examined brain regions and, at least in part, might differentiate behavioural disturbances in adult offspring. Consequently, the future effort to identify the biological background of these interactions in the Poly I:C-induced MIA model in Sprague-Dawley rats is desirable to unequivocally clarify this issue.     

Virus-Based Nanoreactors with GALT Activity for Classic Galactosemia Therapy

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.     

AP2M1 Supports TGF-β Signals to Promote Collagen Expression by Inhibiting Caveolin Expression

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.     

Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts

The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.     

Ablative Radiotherapy Reprograms the Tumor Microenvironment of a Pancreatic Tumor in Favoring the Immune Checkpoint Blockade Therapy

The low overall survival rate of patients with pancreatic cancer has driven research to seek a new therapeutic protocol. Radiotherapy (RT) is frequently an option in the neoadjuvant or palliative settings for pancreatic cancer treatment. This study explored the effect of RT protocols on the tumor microenvironment (TME) and their consequent impact on anti-programmed cell death ligand-1 (PD-L1) therapy. Using a murine orthotopic pancreatic tumor model, UN-KC-6141, RT-disturbed TME was examined by immunohistochemical staining. The results showed that ablative RT is more effective than fractionated RT at recruiting T cells. On the other hand, fractionated RT induces more myeloid-derived suppressor cell infiltration than ablative RT. The RT-disturbed TME presents a higher perfusion rate per vessel. The increase in vessel perfusion is associated with a higher amount of anti-PD-L1 antibody being delivered to the tumor. Animal survival is increased by anti-PD-L1 therapy after ablative RT, with 67% of treated animals surviving more than 30 days after tumor inoculation compared to a median survival time of 16.5 days for the control group. Splenocytes isolated from surviving animals were specifically cytotoxic for UN-KC-6141 cells. We conclude that the ablative RT-induced TME is more suited than conventional RT-induced TME to combination therapy with immune checkpoint blockade.     

Heteroalleles in Common Wheat: Multiple Differences between Allelic Variants of the Gli-B1 Locus

The Gli-B1-encoded γ-gliadins and non-coding γ-gliadin DNA sequences for 15 different alleles of common wheat have been compared using seven tests: electrophoretic mobility (EM) and molecular weight (MW) of the encoded major γ-gliadin, restriction fragment length polymorphism patterns (RFLPs) (three different markers), Gli-B1-γ-gliadin-pseudogene known SNP markers (Single nucleotide polymorphisms) and sequencing the pseudogene GAG56B. It was discovered that encoded γ-gliadins, with contrasting EM, had similar MWs. However, seven allelic variants (designated from I to VII) differed among them in the other six tests: I (alleles Gli-B1i, k, m, o), II (Gli-B1n, q, s), III (Gli-B1b), IV (Gli-B1e, f, g), V (Gli-B1h), VI (Gli-B1d) and VII (Gli-B1a). Allele Gli-B1c (variant VIII) was identical to the alleles from group IV in four of the tests. Some tests might show a fine difference between alleles belonging to the same variant. Our results attest in favor of the independent origin of at least seven variants at the Gli-B1 locus that might originate from deeply diverged genotypes of the donor(s) of the B genome in hexaploid wheat and therefore might be called “heteroallelic”. The donor’s particularities at the Gli-B1 locus might be conserved since that time and decisively contribute to the current high genetic diversity of common wheat.     

Proton Nuclear Magnetic Resonance-Based Method for the Quantification of Epoxidized Methyl Oleate

Epoxidized methyl esters (EMO) with their high oxirane ring reactivity, acts as a raw material in the synthesis of various industrial chemicals including polymers, stabilizers, plasticizers, glycols, polyols, carbonyl compounds, biolubricants etc. EMO has been generally quantified by the gas chromatography (GC) and high-performance liquid chromatography (HPLC) techniques. Taking into the account of the limitations of these techniques, two qHNMR-based equations have been proposed for the quantification of EMO in the mixture of EMO and methylesters (MO). The validity of the proposed method was determined using standard mixtures of MO and EMO having different molar concentrations. The developed equations have been applied on the samples of EMO prepared from oleic acid in two-step process viz., esterification followed by epoxidation. The qHNMR-based EMO quantification showed acceptable agreement with the results obtained from HPLC analysis.     

Borage Oil Enhances Lamellar Body Content and Alters Fatty Acid Composition of Epidermal Ceramides in Essential Fatty Acid-Deficient Guinea Pigs

Borage oil [BO: 40.9% linoleic acid (LNA) and 24.0% γ-linolenic acid (GLA)] reverses disrupted epidermal lipid barrier in essential fatty acid deficiency (EFAD). We determined the effects of BO on lamellar body (LB) content and LNA and GLA incorporation into epidermal ceramide 1 (CER1) and epidermal ceramide 2 (CER2), major barrier lipids. EFAD was induced in guinea pigs by a diet of 6% hydrogenated coconut oil (HCO) for 10 weeks (group HCO) or 8 weeks followed by 6% BO for 2 weeks (group HCO + BO). LB content and LNA and GLA incorporation into CER1 were higher in group HCO + BO than in group HCO. Small but significant levels of LNA, GLA, and their C20-metabolized fatty acids [dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA)] were incorporated into CER2, where ARA was detected at a level lower than LNA, but DGLA incorporation exceeded that for GLA in group HCO + BO. Dietary BO enhanced LB content and differential incorporation of GLA into CER1 and DGLA into CER2.     

Characterization of Dog Glutathione Transferase P1-1, an Enzyme Relevant to Veterinary Medicine

Glutathione transferases (GSTs) form a family of detoxication enzymes instrumental in the inactivation and elimination of electrophilic mutagenic and carcinogenic compounds. The Pi class GST P1-1 is present in most tissues and is commonly overexpressed in neoplastic cells. GST P1-1 in the dog, Canis lupus familiaris, has merits as a marker for tumors and as a target for enzyme-activated prodrugs. We produced the canine enzyme CluGST P1-1 by heterologous bacterial expression and verified its cross-reactivity with antihuman-GST P1-1 antibodies. The catalytic activity with alternative substrates of biological significance was determined, and the most active substrate found was benzyl isothiocyanate. Among established GST inhibitors, Cibacron Blue showed positive cooperativity with an IC₅₀ value of 43 nM. Dog GST P1-1 catalyzes activation of the prodrug Telcyta, but the activity is significantly lower than that of the human homolog.