Immunoreactive Protein Research Articles

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Fuzapladib in a randomized controlled multicenter masked study in dogs with presumptive acute onset pancreatitis.

Currently, no specific treatment is available for acute onset pancreatitis (AP), and management relies on symptomatic and supportive standard of care (SOC). Fuzapladib is a novel leukocyte function-associated antigen type-1 (LFA-1) activation inhibitor, blocking activation and subsequent adhesion and migration of neutrophils, potentially decreasing the risk of pancreatitis progression and systemic inflammation. Evaluate the safety and clinical response of dogs with AP after 3 days of administration of fuzapladib. Sixty-one client-owned dogs with presumptive AP. Randomized, masked, and placebo controlled multicenter study. Sixty-one dogs with AP were included for safety assessment, whereas 35 evaluable cases (fuzapladib, n = 16; placebo, n = 19) were included for clinical evaluation. Clinical improvement was assessed based on the change in the modified clinical activity index (MCAI) score on Day 3 compared to Day 0. Secondary variables included canine acute pancreatitis clinical severity index (CAPCSI) scores and serum concentrations of canine pancreatic lipase immunoreactivity, cytokines, and C-reactive protein. Fuzapladib was well tolerated by all treated dogs. Mean change in MCAI scores was significantly higher in the fuzapladib-treated (-7.75) than the placebo group (-5.68; P = .02, 95% confidence interval [CI] for the difference, -4.33, -0.35), suggesting clinical improvement in fuzapladib-treated dogs. No significant difference was found in any of the secondary variables between groups. Administration of fuzapladib to dogs was safe, and a favorable response was detected in 2 clinical activity scores. Effects of fuzapladib on survival and duration of hospitalization were not studied.

Biotransformation of wheat proteins with Lactobacillus sp. following high hydrostatic pressure pre-treatment to reduce gluten immunoreactivity

The effect of high hydrostatic pressure (HHP, 50–300 MPa, 5 min, 18 °C) on the biotransformation of wheat proteins with Lactobacillus acidophilus or L. sanfranciscensis was studied. Increasing the pressure from 50 to 150 MPa before sourdough fermentation did not change the size of the bacterial population, but it increased the endoproteolytic activity of Lactobacillus enzymes, degree of protein hydrolysis and decreased the concentration of gliadins and gluten immunoreactivity. Fermentation after pressurization at 150 MPa reduces to 44.0 ± 0.1 and 25.6 ± 1.1%, the immunoreactivity of proteins in sourdough obtained by fermentation with L. acidophilus and L. sanfranciscensis, respectively. Further increasing the pressure from 150 to 300 MPa resulted in the reduction of bacterial growth, reduction of endoproteolytic activity, degree of protein hydrolysis, gliadin degradation and gluten immunoreactivity. Sourdough treatment at 250 and 300 MPa increased the antigen concentration compared to the control samples. The fermentation of sourdough with L. acidophilus and L. sanfranciscensis, following increasing pressurization, resulted in an increase in the β-sheets and a decrease in the α-helix structure of the protein. Applying the pressure of 250 and 300 MPa changes the conformation of gluten, and reduced significantly activity of peptidases of Lactobacillus strains, thereby increasing protein immunoreactivity.

WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

VEGF-A ameliorates ischemia hippocampal neural injury via regulating autophagy and Akt/CREB signaling in a rat model of chronic cerebral hypoperfusion

ObjectiveChronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear. MethodsTo address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively. ResultsLC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693. ConclusionPresent results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.

Helicobacter pylori strains and their relationship with vacuolating cytotoxin A gene in the increased risk of gastric cancer

ObjectivesThe main objective of this study is to analyze the relationship of H. pylori vacuolating gene in gastric cancer. MethodsH. pyroli strains were isolated from the biopsy specimens collected from 50 patients suspected of gastritis, gastric carcinoma and peptic ulcer disease. H. pylori JK was identified and the virulence gene was determined. The presence of vacuolating cytotoxin A gene was amplified using a polymerase chain reaction. The growth kinetics of the strain JK were evaluated up to 96 h at 37 °C and the culture was used for the determination of VacA gene expression. ResultsThe screened H. pylori JK was positive for vacA gene and showed heterogeneity among H. pylori. Bacterial growth increased up to 48 h incubation and declined after 48 h. The isolated strain showed maximum growth after 72 h incubation, whereas, the toxic protein reached maximum yield after 96 h incubation and was not associated with growth. It was resistant to antibiotics such as ceftriaxone, cefuroxime, cefalexin, and co-trimoxazole. The intracellular H. pylori culture extracts induced vacuolation in HeLa cell lines in vitro. The isolated H. pylori strain showed more than 43% vacuolating activity after 24 h incubation. Immunoblotting assay revealed the presence of an immuno-reactive protein band in the culture supernatant of H. pylori JK. These results indicated the pathogenic activity of the VacA gene and toxin production. ConclusionsGenerally, H. pylori persists for the lifetime of the individual, revealing the significance of eradication from the host.

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, β, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Beta-caryophyllene inhibits the permeability of the blood–brain barrier in MPTP-induced parkinsonism

IntroductionParkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide. Although the precise pathogenesis of PD remains unclear, several studies demonstrate that oxidative stress, inflammation, low levels of antioxidants, and the presence of biomolecules that generate reactive oxygen species can disrupt the blood–brain barrier (BBB) as an essential feature of the disease. AimsThis study aimed to test whether agonism to cannabinoid receptor type 2 (CB2) through the administration of β-caryophyllene (BCP) could correct BBB permeability in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonism induction model. MethodsWe conducted a molecular assessment of proteins (immunochemistry and western blot), BBB permeability, and related biomarkers of PD (lipid peroxidation) in the MPTP mouse model of the disease. ResultsExpression of zonula occludens (ZO-1) and occludin tight junction (TJ) proteins was dampened in the striatum and substantia nigra pars compacta of mice, while lipid peroxidation and BBB permeability increased in the striatum in the MPTP-treated group, and these effects were reversed under BCP administration. This phytocannabinoid was able to restore protein expression and immunoreactivity of tyrosine hydroxylase (TH), ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP), as well as nuclear factor-erythroid 2-related factor (NRF2) translocation to the nucleus, and NADPH quinone oxidase 1 (NQO1) expression in mice treated with MPTP. ConclusionThese results highlight the role of CB2 as a therapeutic target for PD, suggesting that its activation may ameliorate PD-related BBB disruption and oxidative stress, reducing the selective death of dopaminergic neurons.

Altered Expression of Heme Oxygenase 2 in Heme Oxygenase 1-deficient Mouse Embryos.

Heme oxygenases (Hmoxs) are enzymes that catalyze the first and rate-limiting step in the degradation of heme to carbon monoxide, iron, and biliverdin. The two main isozymes, namely Hmox1 and Hmox2, are encoded by two different genes. Mutation of the Hmox1 gene in mice is known to cause extensive prenatal lethality, and limited information is available about the expression of Hmox proteins in developing mouse embryos. In this study, immunohistochemistry was used to perform a detailed investigation comparing Hmox proteins in Hmox1 wild-type and knockout (KO) mouse embryos collected from wild-type and heterozygous timed-matings. Western analysis for Hmoxs was also done in the organs of late-gestation embryos. The results demonstrated cytoplasmic and nuclear localization of Hmoxs in all the organs examined in wild-type embryos. Interestingly, Hmox2 immunoreactive protein signals were significantly low in most of the organs of mid- and late-gestation Hmox1-KO embryos. Furthermore, relative levels of Hmox2 were revealed to be significantly lower in the lung and kidney of late-gestation Hmox1-KO embryos by western analysis, which complemented the immunohistochemistry findings in these two organs. The current study provides detailed immunoexpression patterns of Hmox proteins in wild-type and Hmox1-KO mouse embryos in mid- and late-gestation.

Therapeutic effect of thymoquinone on brain damage caused by nonylphenol exposure in rats.

Nonylphenol (NP), causes various harmful effects such as cognitive impairment and neurotoxicity. Thymoquinone (TQ), has antioxidant, anti-inflammatory, and neuroprotective properties. In this study, our aim is to investigate the effects of TQ on the brain damage caused by NP. Corn oil was applied to the control group. NP (100 mg/kg/day) was administered to the NP and NP + TQ groups for 21 days. TQ (5 mg/kg/day) was administered to the NP + TQ and TQ groups for 7 after 21 days. At the end of the experiment, the new object recognition test was applied to the rats and the rats were killed and their brain tissues were removed. Sections taken from brain tissues were stained with hematoxylin-eosin for histopathological evaluation. In addition, neuronal nuclei (NeuN), glial fibrillary acidic protein(GFAP), Cas-3, and nervegrowth factor (NGF) immunoreactivities were evaluated in brain tissue sections. In addition,malondialdehyde (MDA), superoxide dismutase(SOD), and catalase(CAT) activities were determined. Comet assay was applied to determine DNA damage in cells. The results of our study showed that NP, caused behavioral disorders and damage to the cerebral cortex in rats. This damage in the form of neuron degeneration seen in the cortex was associated with apoptosis involving Cas-3 activation, increased DNA damage, and free oxygen radicals. NP, SOD, and CAT caused a decrease in enzyme activities. In addition, the cellular protein NeuN was decreased, astrocytosis-associated GFAP was increased, and growth factor NGF was decreased. When all our evaluations are taken together, treatment with TQ showed an ameliorative effect on the behavioral impairment and brain damage caused by NP exposure.

Novel NONO::TFE3 fusion and ALK co-expression identified in a subset of cutaneous microcystic/reticular schwannoma.

Microcystic/reticular schwannoma (MRS) is a benign variant of schwannoma with a predilection for the gastrointestinal tract and skin. To date, genetic characterization of this tumor is limited. Prompted by the identification of TFE3::NONO fusion and ALK overexpression in an index case of MRS, a cohort of tumors was collected from institutional and consultation archives of two institutions. Next-generation sequencing (NGS), TFE3 fluorescence in situ hybridization (FISH), and TFE3 and ALK immunohistochemistry were performed, while clinicopathologic variables were documented. Eighteen MRS cases were identified (35 to 85 years) arising in the skin (n=8), gastrointestinal tract (n=5), adrenal gland (n=3), abdominal wall (n=1), and unknown site (n=1). Tumors showed a circumscribed to multinodular to plexiform low-power architecture with variable amounts of microcystic/reticular and solid schwannian components. Mitotic figures were scarce (0-1/10 HPFs), and atypia was absent. S100 protein and/or SOX10 immunoreactivity was noted in the microcystic/reticular and schwannian areas of all cases. NGS performed on two cutaneous tumors yielded NONO exon 12 fusion with TFE3 exon 4, and these lesions also showed HMB45 and ALK expression. Two additional cases showed ALK expression (1 weak), while a third was positive for TFE3, but these cases failed to show ALK or TFE3 rearrangement by FISH/NGS. There were no morphologic variables that correlated with the presence of NONO::TFE3. We identified a subset of microcystic/reticular schwannomas with NONO::TFE3 fusions and ALK co-expression, adding to the cohort of mesenchymal neoplasms that show ALK overexpression without rearrangement of the ALK gene.

Modulation of soy protein immunoreactivity by different matrix structures of lactic acid bacterium-induced soy protein gels: Epitope destruction during in vitro gastroduodenal digestion and absorption

Soy allergy is a common health problem. Food structure may change the gastroduodenal digestion and absorption of soy proteins, thus leading to the modulation of the immunoreactivity of soy proteins. In this study, lactic acid bacterium (LAB)-fermented soy protein isolates (FSPIs) were prepared at four concentrations (0.2 %–5.0 %, w/v) to present various matrix structures (nongel, NG; weak gel, WG; medium gel, MG; and firm gel, FG) and subjected to in vitro dynamic gastroduodenal digestion model. The results of sandwich enzyme-linked immunosorbent and human serum IgE binding capacity assays demonstrated that FSPI gels, especially the FSPI-MG/WG digestates obtained at the early and medium stages of duodenal digestion (D-5 and D-30), possessed greater potency in immunoreactivity reduction than FSPI-NG and reduced to 1.9 %–68.3 %. The transepithelial transport study revealed that the immunoreactivity of FSPI-MG/WG D-5 and D-30 digestates decreased through the stimulation of interferon-γ production and the induction of dominant Th1/Th2 differentiation. Peptidomics and bioinformatics analyses illustrated that compared with FSPI-NG, the FSPI-gel structure promoted the epitope degradation of the major allergens glycinin G2/G5, β-conglycinin α/β subunit, P34, lectin, trypsin inhibitor, and basic 7S globulin. Spatial structure analysis showed that FSPI-gel elicited an overall promotion in the degradation of allergen epitopes located in interior and exterior regions and was dominated by α-helix and β-sheet secondary structures, whereas FSPI-MG/WG promoted the degradation of epitopes located in the interior region of glycinin/β-conglycinin and exterior region of P34/basic 7S globulin. This study suggested that the FSPI-gel structure is a promising food matrix for decreasing the allergenic potential of allergenic epitopes during gastroduodenal digestion and provided basic information on the production of hypoallergenic soy products.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.

Brucellosis detection and the role of Brucella spp. cell wall proteins.

Brucellosis remains an endemic zoonotic disease in many developing countries, causing great harm to public health and devastating losses to livestock. One of the main reasons for the low effectiveness of anti-brucellosis measures is the lack of reliable methods for diagnosing infected animals throughout their lifespan. Classical serological tests, such as the tube agglutination test, rose Bengal plate test, and complement fixation test, as well as commercial enzyme-linked immunosorbent assay kits, are based on the detection of antibodies to the cell wall polysaccharide antigens of Brucella spp. smooth strains. As a result, they do not exclude cross-reactions with related bacteria and fail to differentiate between infected and vaccinated animals. Over the past decades, many attempts have been made to identify immunoreactive and pathogen-specific protein antigens. To date, several studies have investigated Brucella spp. recombinant proteins, including cell wall proteins, as the best antigens for diagnosing brucellosis in animals and humans. However, the available results on the specificity and sensitivity of serological tests based on cell wall proteins are ambiguous and sometimes contradictory. This review aims to provide an overview of the current state of knowledge of the diagnostic value of outer membrane and/or periplasmic proteins of Brucella spp. The goal is to identify future developments that may lead to reliable antigens for serological tests.

Evaluating the effect of post-traumatic hypoxia on the development of axonal injury following traumatic brain injury in sheep

Damage to the axonal white matter tracts within the brain is a key cause of neurological impairment and long-term disability following traumatic brain injury (TBI). Understanding how axonal injury develops following TBI requires gyrencephalic models that undergo shear strain and tissue deformation similar to the clinical situation and investigation of the effects of post-injury insults like hypoxia. The aim of this study was to determine the effect of post-traumatic hypoxia on axonal injury and inflammation in a sheep model of TBI. Fourteen male Merino sheep were allocated to receive a single TBI via a modified humane captive bolt animal stunner, or sham surgery, followed by either a 15 min period of hypoxia or maintenance of normoxia. Head kinematics were measured in injured animals. Brains were assessed for axonal damage, microglia and astrocyte accumulation and inflammatory cytokine expression at 4 hrs following injury. Early axonal injury was characterised by calpain activation, with significantly increased SNTF immunoreactivity, a proteolytic fragment of alpha-II spectrin, but not with impaired axonal transport, as measured by amyloid precursor protein (APP) immunoreactivity. Early axonal injury was associated with an increase in GFAP levels within the CSF, but not with increases in IBA1 or GFAP+ve cells, nor in levels of TNFα, IL1β or IL6 within the cerebrospinal fluid or white matter. No additive effect of post-injury hypoxia was noted on axonal injury or inflammation. This study provides further support that axonal injury post-TBI is driven by different pathophysiological mechanisms, and detection requires specific markers targeting multiple injury mechanisms. Treatment may also need to be tailored for injury severity and timing post-injury to target the correct injury pathway.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3β at Ser9 (inactive) followed by PGC-1β accumulation. While selective inhibition of GSK3β promoted PGC-1β, Akt inhibition could abrogate IGF1-induced p-GSK3β (Ser9) and PGC-1β immunoreactive protein indicating Akt-mediated GSK3β inactivation and PGC-1β stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

Evidence for the expression of vasorin in the human female reproductive tissues

Objective: To investigate the expression and localization of Vasorin (Vasn) in human female reproductive system. Methods: The presence of Vasorin was evaluated by RT-PCR and immunoblotting analyses in patient-derived endometrial, myometrial and granulosa cells (GCs) primary cultures. Immunostaining analyses were performed to detect Vasn localization in primary cultures and in ovarian and uterine tissues. Results: Vasn mRNA was detected in patient-derived endometrial, myometrial and GCs primary cultures without significant differences at the transcript level. Otherwise, immunoblotting analysis showed that Vasn protein levels were significantly higher in GCs than proliferative endometrial stromal cells (ESCs) and myometrial cells. Immunohistochemistry performed in ovarian tissues revealed that Vasn was expressed in the GCs of ovarian follicles at different stages of development with a higher immunostaining signal in mature ovarian follicles such as the antral follicle or on the surface of cumulus oophorus cells than in early-stage follicles. The immunostaining of uterine tissues showed that Vasn was expressed in the proliferative stroma endometrium while it was significantly less expressed in the secretory endometrium. Conversely, no protein immunoreactivity was revealed in health myometrial tissue. Conclusions: Our results revealed the presence of Vasn in the ovary and the endometrium. The pattern of Vasn expression and distribution suggests that this protein may have a role in the regulation of processes such as folliculogenesis, oocyte maturation, and endometrial proliferation.

Translocation of Methionine Adenosyl Transferase MAT2A and Its Prognostic Relevance for Liver Hepatocellular Carcinoma.

Methionine adenosyl transferases (MATs) catalyze the synthesis of the biological methyl donor adenosylmethionine (SAM). Dysregulation of MATs has been associated with carcinogenesis in humans. We previously found that downregulation of the MAT1A gene enriches the protein-associated translation process and worsens liver hepatocellular carcinoma (LIHC) prognosis. We also discovered that subcellular localization of the MAT2A protein has independently prognostic relevance in breast cancer patients. The present study aimed to examined the clinical relevance of MAT2A translocation in human LIHC. Essential methionine cycle gene expressions in TCGA LIHC datasets were analyzed using Gene Expression Profiling Interactive Analysis 2 (GEPIA2). The protein expression pattern of MAT2A was determined in the tissue array of our own LIHC cohort (n = 261) using immuno-histochemistry, and the prognostic relevance of MAT2A protein's subcellular localization expression was examined using Kaplan-Meier survival curves. LIHC patients with higher MAT2A mRNA expression had a worse survival rate (p = 0.0083). MAT2A protein immunoreactivity was observed in both cytoplasm and nucleus fractions in the tissue array. Tumor tissues had elevated MAT2A protein expression in both cytoplasm and nucleus compared to their adjacent normal tissues. A higher cytoplasmic to nuclear MAT2A protein expression ratio (C/N) was found in female LIHC patients compared to that of male patients (p = 0.047). Kaplan-Meier survival curves showed that a lower MAT2A C/N correlated with poor overall survival in female LIHC patients (10-year survival rate: 29.2% vs. 68.8%, C/N ≤ 1.0 vs. C/N > 1.0, log-rank p = 0.004). Moreover, we found that specificity protein 1 (SP1) may have a potential interaction with nuclear MAT2A protein, using protein-protein interaction; this we found using the GeneMANIA algorithm. We explored the possible protective effects of the estrogen axis in LIHC using the Human Protein Atlas (HPA), and found evidence supporting a possible protective effect of estrogen-related protein ESSRG in LIHC. The localization of SP1 and MAT2 appeared to be inversely associated with ESRRG expression in LIHC. The present study demonstrated the translocation of MAT2A and its prognostic relevance in female LIHC patients. Our findings suggest the potential of estrogen in SP1 regulation and localization of MAT2A, as therapeutic modalities against in female LIHC patients.

Type 1 secretion system and effectors in Rickettsiales.

Obligate intracellular bacteria in the order Rickettsiales are transmitted by arthropod vectors and cause life-threatening infections in humans and animals. While both type 1 and type 4 secretion systems (T1SS and T4SS) have been identified in this group, the most extensive studies of Rickettsiales T1SS and associated effectors have been performed in Ehrlichia. These studies have uncovered important roles for the T1SS effectors in pathobiology and immunity. To evade innate immune responses and promote intracellular survival, Ehrlichia and other related obligate pathogens secrete multiple T1SS effectors which interact with a diverse network of host targets associated with essential cellular processes. T1SS effectors have multiple functional activities during infection including acting as nucleomodulins and ligand mimetics that activate evolutionarily conserved cellular signaling pathways. In Ehrlichia, an array of newly defined major immunoreactive proteins have been identified that are predicted as T1SS substrates and have conformation-dependent antibody epitopes. These findings highlight the underappreciated and largely uncharacterized roles of T1SS effector proteins in pathobiology and immunity. This review summarizes current knowledge regarding roles of T1SS effectors in Rickettsiales members during infection and explores newly identified immunoreactive proteins as potential T1SS substrates and targets of a protective host immune response.

Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.