Immunolocalization Of Protein Research Articles

Spatial proteomics of Onchocerca volvulus with pleomorphic neoplasms shows local and systemic dysregulation of protein expression.

Onchocerca volvulus , the causative agent of onchocerciasis (river blindness), is targeted for elimination by WHO. The primary strategy involves mass administration of ivermectin. A small proportion of adult female worms develop pleomorphic neoplasms (PN). Here, we used laser capture microdissection and highly sensitive mass spectrometry analysis to determine the protein inventory of PN to identify proteins that may be associated with tumor development. Neoplasm tissue from female worms was analyzed, and compared to normal tissue from the body wall, uterus and intestine from the same worms, and to tissues from females without PN. When compared, PN and healthy control (HC) worms display a different set of proteins, the PN tissue being the one with the highest number of proteins (1,390). From these, 594 were not present in any HC worm tissue. Despite the large number of proteins identified in PN tissue, their low abundance suggests also in PN dysregulation of protein expression. Immunolocalization of a calcium binding protein detected in PN confirmed the mass spectrometry results. In conclusion, we have developed a system to analyze the proteome of O. volvulus from nodule sections and identified proteins that are potentially linked to the development of PN and may contribute to worm mortality.

Creation of a novel CRISPR-generated allele to express HA epitope-tagged C1QL1 and improved methods for its detection at synapses.

C1QL1 is expressed in a subset of cells in the brain and likely has pleiotropic functions, including the regulation of neuron-to-neuron synapses. Research progress on C1QL proteins has been slowed by a dearth of available antibodies. Therefore, we created a novel knock-in mouse line in which an HA-tag is inserted into the endogenous C1ql1 locus. We examined the entire brain, identifying previously unappreciated nuclei expressing C1QL1, presumably in neurons. By total numbers, however, the large majority of C1QL1-expressing cells are of the oligodendrocyte lineage. Subcellular immunolocalization of synaptic cleft proteins is challenging, so we developed a new protocol to improve signal at synapses. Lastly, we compared various anti-HA antibodies to assist future investigations using this and likely other HA epitope-tagged alleles.

Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + .The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences.Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.

The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.

Alterations in immunolocalisation of non-collagenous proteins in cartilage and bone tissue after gastrectomy, antrectomy and fundectomy in rat model

Bone, as a tissue, plays a pivotal role in maintaining whole body homeostasis, contributing to, among other things, structural support, blood cell production, and mineral storage. On the other hand, bone homeostasis depends on hormonal factors released by various tissues of the body, including the gastrointestinal tract. Thus, surgical procedures involving the removal of various parts of the stomach wall, such as, antrectomy and fundectomy or partial gastrectomy, may have profound negative effects on bone metabolism and mineralization. These effects may result from changes in the concentration of neuropeptides, such as nesfatin-1, secreted by cells located in the fundus and vestibule of the stomach. Additionally, bone morphogenetic proteins, osteocalcin, osteoprotegerin, and tissue inhibitor of metalloproteinases 2, are regulatory proteins involved in bone metabolism, playing a key role in maintaining the balance between the activity of osteoclasts and osteoblasts, cells responsible for the remodeling of bone tissue. The aim of this study was to investigate the effects of antrectomy, fundectomy, and partial gastrectomy, on bone homeostasis in a rat model. The experiment was carried out on 24 male Wistar rats randomly and equally divided into control (SHO), gastrectomy (GAST), antrectomy (ANT), and fundectomy (FUN) groups. At the beginning of the study, the rats underwent surgical removal of selected parts of the stomach. At the end of the 6-week study, the rats were sacrificed, and femurs were collected. Microscopic images of immunohistochemical reactions (IR) in trabecular and compact bone, as well as isolated articular cartilages and growth plates of femurs were analyzed. The intensity of the immunoreaction (IR) of osteocalcin (OC), osteoprotegerin (OPG), tissue inhibitor of metalloproteinases 2 (TIMP-2), bone morphogenetic protein 2 (BMP-2), and nesfatin-1 was determined, and IR-positive cells were counted in each area of the femur. The quantitative analysis of the intensity of OC IR in growth plate cartilage and trabecular bone showed the highest values after fundectomy in comparison to the other treatments. Moreover, fundectomy increased the intensity of BMP-2 IR in articular cartilage, growth plate cartilage, and trabecular bone. The intensity of OPG IR in articular cartilage and compact bone showed the highest values after antrectomy. The intensity of TIMP-2 IR in the examined parts of the femur was highest in the rats subjected to antrectomy. The intensity of expression of nesfatin-1 IR was highest in articular and growth plate cartilages for rats subjected to gastrectomy. In conclusion, surgical procedures affecting the gastrointestinal tract, such as antrectomy, fundectomy, and partial gastrectomy, can have significant effects on bone metabolism. Our study on a rat model demonstrated varying impacts on bone homeostasis, with different surgeries leading to alterations in the expression of key regulatory proteins involved in bone remodeling.

Novel Insights into the Wattle and Daub Model of Entamoeba Cyst Wall Formation and the Importance of Actin Cytoskeleton.

The "Wattle and Daub" model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation.

Immunolocalization of Pectins and Arabinogalactan Proteins in Young Ovules of Selected Amphimictic and Apomictic Species of the Asteraceae Family

Immunolocalization of Pectins and Arabinogalactan Proteins in Young Ovules of Selected Amphimictic and Apomictic Species of the Asteraceae Family

Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P< 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P< 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.

Morphometrical and Immunohistochemical Evaluation of Kidney as an Indirect Parameter to Estimate Age in Puppies in Veterinary Forensic Pathology.

Estimation of age represents a central focus in the veterinary forensic pathology field. Currently, the visual examination of the dentition and the skeletal age are the main methods to estimate the age of puppies. Nevertheless, these methods are affected by a broad range of variables. In contrast, the kidney is characterized by a specific postnatal development. In human glomerulogenesis, fetal mesangial cells change their immunohistochemical phenotypes with maturation. Therefore, we hypothesized that histological and immunohistochemical examinations of the kidney can be used together as an indirect parameter for age determination in puppies' cadavers. Forty-five puppies' cadavers were divided into five groups defined by age (Group A= 0-15 days, Group B = 16-45 days, Group C = 46-85 days, Group D = 86-105 days, Group E= 105-365 days). For each case, kidney samples were collected and processed for histopathological (for morphometrical study of the glomerulus) and immunohistochemical (for the immunolocalization of the α-SMA protein) studies. Morphometrical study allowed us to observe statistical differences in the mean glomerulus numbers per field among assessed groups. Similarly, immunohistochemical examination showed differences in SMA expression among groups. Our findings suggest a potential use of kidney morphometrical and immunohistochemical examinations together as an indirect parameter to assess the age of illegally imported puppies.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.)

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor β superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

Immunolocalization and expression of Siglec5 protein in the male reproductive tract of dromedary camel during rutting season.

Lectins are carbohydrate-binding proteins that are highly selective for sugar groups on other molecules. Siglec5 is a cell-surface lectin that belongs to the sialic acid-binding Ig-like lectins (Siglecs) and acts as a suppressor of immune responses. In this study, immunohistochemistry, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of Siglec5 in the male reproductive tract of dromedary camels during the rutting season. Siglec5 displayed strong immunostaining in the cranial and caudal testicular regions and moderate immunostaining in the rete testis. Different parts of the epididymis showed varying immunoreactions to Siglec5. The spermatozoa in the testes and epididymis also showed positive immunostaining for Siglec5, whereas, the vas deferens showed negative immunostaining for the protein. The results obtained by western blotting confirmed the immunohistochemical detection of the protein in the testicular and epididymal tissues. The results of qRT-PCR showed that Siglec mRNA was expressed differently in each part of the testis and epididymis; the highest levels of expression were observed in the caudal part of the testis and in the head of the epididymis. In conclusion, the present study revealed that Siglec5 is mainly located in the testis and epididymis, where sperm production and maturation occur. Therefore, this protein may play an essential role in the development, maturation and protection of camel sperm.

Exposure to Dichlorvos pesticide alters the morphology of and lipid metabolism in the ventral prostate of rats.

Organophosphate pesticides are widely used in agriculture, leading to soil, water, and food contamination. Among these compounds is Dichlorvos [O,O-dimethyl O-(2,2-dichlorovinyl)phosphate, DDVP], which is listed as a highly toxic compound by the Environmental Protection Agency and World Health Organization. Exposure to DDVP can result in nervous, respiratory, hepatic, and reproductive abnormalities, in addition to endocrine disrupting, mutagenic, and carcinogenic effects. Little is known about the impacts of DDVP on the reprogramming of lipid metabolism, which is also associated with the development and progression of cancer, since the tumor cells need to recruit, capture, and use fatty acids to compose their building membranes. This study aimed to evaluate the influence of the pesticide DDVP on lipid metabolism in the prostate, after chemical induction by the carcinogen N-methyl-N-nitrosourea (MNU). For this, 32 Fischer rats aged 90days were randomly divided into four experimental groups: Control, DDVP, MNU, and MNU + DDVP. The MNU and MNU + DDVP groups underwent chemical induction with MNU (15mg/kg) and the DDVP and MNU + DDVP groups received a diet supplemented with DDVP (10mg/kg). Histopathological analyses of the rat ventral prostate showed 100% incidence of epithelial hyperplasia in the MNU and MNU + DDVP groups. This finding was accompanied by an increase of the epithelial compartment in the MNU + DDVP group. Immunolocalization of important proteins linked to lipid metabolism has been established. In the MNU + DDVP group, Western blotting analyses pointed out an increased expression of the protein LIMP II (Lysosomal Integral Membrane Protein-II), which is correlated with the capture and distribution of lipids in tumor cells. Together, these results indicate that the association of a low dose of DDVP with MNU was able to promote alterations in the morphology and lipid metabolism of the rat ventral prostate, which may be related to tumor progression in this organ.

Immunolocalization of Desmin, Vimentin and S-100 proteins in testis and epididymis of African striped ground squirrel (Xerus erythropus)

The aim of the study was to immunolocalize desmin, vimentin and S-100 proteins in the testes and epididymis of African striped ground squirrel (Xerus erythropus) and establish their spatial distribution in the two organs. Formalin fixed-paraffin-embedded sections of the testis and epididymis obtained from ten apparently healthy adult male African striped ground squirrels were processed routinely for immunohistochemistry, using primary antibodies specific to desmin, vimentin and S-100. S-100The results showed that desmin reacted intensely in the myoid cells of the seminiferous tubules and smooth muscle cells of the epididymal ducts. It showed a moderate positive immunoreaction in the interstitial cells of Leydig, and the epididymal inter-ductal loose connective tissue. However, there was no immunoreaction of desmin in the spermatogonia or any other spermatogenic cell of the seminiferous tubule. Vimentin reacted intensely in the Leydig cells and spermatogonia. It showed moderate positive reaction in the myoid cells and the epididymal inter-ductal loose connective tissue. There was no immunoreaction of vimentin in the other spermatogenic cells (other than the spermatogonia). The S-100 proteins expressed a mild positive immunoreaction at the interstitial cells of Leydig, but negative immunoreaction in all other parts of testis and epididymis. Also, there was intense immunoreaction of desmin and vimentin and moderate immunoreaction of S-100 in the vascular endothelium in the testis and epididymis. In conclusion, the spatial distribution of desmin, vimentin and S-100 proteins in the testes and epididymis in the African striped ground squirrel showed some similarities and contrast with other mammals, giving insight into the functions of the proteins in these organs of the rodent.

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment. Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

Follicular Atresia, Cell Proliferation, and Anti-Mullerian Hormone in Two Neotropical Primates (Aotus nancymae and Sapajus macrocephalus).

This study evaluated the follicular atresia, cell proliferation, and anti-Mullerian hormone action in Aotus nancymae and Sapajus macrocephalus during three sexual phases (follicular, luteal, and gestational). Follicular quantification and immunolocalization of Caspase-3 protein, B-cell lymphoma 2 (BCL-2), proliferating cell nuclear antigen (PCNA), and anti-Mullerian hormone (AMH) were performed. A significant difference in the quantification between preantral and antral follicles, with a progressive decrease in the antrals, was identified. Protein and hormonal markers varied significantly between follicle cell types (A. nancymae p = 0.001; S. macrocephalus, p = 0.002). Immunostaining in the preantral and antral follicles was present in all sexual phases; for Caspase-3, in granulosa cells, oocytes, and stroma; for BCL-2, in granulosa cells, oocytes, and theca; and for PCNA and AMH, in oocytes and granulosa cells. The immunostaining for Caspase-3 was more expressive in the preantral follicles (follicular phase, p < 0.05), while that for BCL-2 and PCNA was more expressive in the antral follicles of the follicular phase. The AMH was more expressive in the primary and antral follicles of nonpregnant females, in both the follicular and luteal phases. Our results contribute to understanding the ovarian follicular selection, recruitment, and degeneration of these species.

Immunolocalization of Pglyrp3 and Eps8l1 proteins in the regenerating lizard epidermis indicates they contribute to epidermal barrier formation

During tail regeneration in lizards the new corneous layer formed in the regenerating epidermis includes antimicrobial peptides, cystatin and serpins, likely forming an anti-microbial barrier. The present study aims to reveal other proteins potentially contributing to this protective barrier of the epidermis. Using immunohistochemistry we have detected a peptidoglycan-like recognition protein-3 (pglyrp3), an antimicrobial molecule, and an epidermal growth factor receptor kinase 8 l (eps8l), a receptor of EGF (Epidermal Growth Factor) that stimulates epidermal formation. The study shows that the two proteins are mostly accumulated in the forming wound epidermis and in the shedding layer of the regenerating scales. The shedding layer is the intra-epidermal layer that allows the separation of the initial corneous layer from the regenerating epidermis. While presence of pglyrp3 is likely related to the formation of the anti-microbial barrier, the function of the eps8l protein in epidermal regeneration remains unknown. Whether the latter protein is involved in keratinocyte movement within the regenerating epidermis has to be specifically determined in future studies. Together with the antimicrobial peptides cystatin and serpins, previously detected in the wound epidermis and shedding layer, the present study indicates that pglyp3, and potentially eps8l, contribute to protect the new skin and underlying regenerated tissues from the potential microbe invasion.

Role of reactive oxygen species in the modulation of auxin flux and root development in Arabidopsis thaliana.

Reactive oxygen species (ROS) play a dual role in plant biology, acting as important signal transduction molecules and as toxic byproducts of aerobic metabolism that accumulate in cells upon exposure to different stressors and lead to cell death. In plants, root architecture is regulated by the distribution and intercellular flow of the phytohormone auxin. In this study, we identified ROS as an important modulator of auxin distribution and response in the root. ROS production is necessary for root growth, proper tissue patterning, cell growth, and lateral root (LR) induction. Alterations in ROS balance led to altered auxin distribution and response in SOD and RHD2 loss-of-function mutants. Treatment of Arabidopsis seedlings with additional sources of ROS (hydrogen peroxide) or an ROS production inhibitor (diphenylene iodonium) induced phenocopies of the mutants studied. Simultaneous application of auxin and ROS increased LR primordia induction, and PIN-FORMED protein immunolocalization further demonstrated the existing link between auxin and ROS in orchestrating cell division and auxin flux during root development. In Arabidopsis roots, genetic alterations in ROS balance led to defective auxin distribution and growth-related responses in roots. Exogenous hydrogen peroxide alters the establishment of the endogenous auxin gradient in the root meristem through regulation of PIN-FORMED polarity, while the simultaneous application of hydrogen peroxide and auxin enhanced LR induction in a dose- and position-dependent manner through activation of cell division.

The Effect of Supplementation with β-Hydroxy-β-Methylbutyric Acid (HMB) to Pregnant Sows on the Mucosal Structure, Immunolocalization of Intestinal Barrier Proteins, VIP and Leptin in the Large Intestine in their Offspring

Abstract The large intestine epithelium plays an important role in water absorption and participates in fluid, acid-base and electrolyte balance, and the removal of waste products. The large intestine is rich in microorganism-presented enzyme activity. Apart from energy supply, the colon also participates in the synthesis of trophic factors and the modulation of the immune system and the systemic inflammatory response. The current study investigated the effects of dietary HMB administration to pregnant sows on the postnatal development of the colon in their offspring, at weaning. From the 70th to the 90th day of gestation, sows received either a basal diet (n = 12) or the basal diet supplemented with HMB (n = 12) at a dose of 0.2 g/kg of body weight/day. Maternal HMB treatment increased serum IgG and glucose concentrations and decreased serum urea concentration in the piglets. Basal histomorphometric analysis of offspring large intestines showed that prenatal HMB treatment led to a reduction in the thickness of the mucosa, submucosa and both types of myenterons, as well as reduced crypt thickness. The immunoreaction performed to mark T0 lymphocytes and total T lymphocytes in the colon wall showed that prenatal HMB treatment decreased the number of both types of lymphocytes. Greater expression for cadherin was found in the colon of piglets delivered by the HMB-treated sows. The expression of both tight junction proteins (occludin and claudin-3), as well as that of leptin, was stronger in the HMB-treated group. Vasoactive intestinal peptide (VIP) expression was stronger in the submucosal plexuses in the HMB maternal treated piglets, while no changes were observed in the myenteric plexuses. The results obtained indicate that the administration of HMB to pregnant sows significantly influenced the expression of leptin, VIP and some proteins of the intestinal barrier in their offspring, with less influence on large intestine basal morphology.

Immunolocalization of pectins and arabinogalactan proteins during fruit abscission in olive (Olea europaea L.)

ISHS XXXI International Horticultural Congress (IHC2022): International Symposium on Integrative Approaches to Product Quality in Fruits and Vegetables Immunolocalization of pectins and arabinogalactan proteins during fruit abscission in olive (Olea europaea L.)