Histochemical Staining Research Articles

Exosomes from umbilical cord mesenchymal stem cells ameliorate intervertebral disc degeneration via repairing mitochondrial dysfunction

BackgroundReactive oxygen species (ROS), predominantly generated by mitochondria, play a crucial role in the pathogenesis of intervertebral disc degeneration (IVDD). Reduction of ROS levels may be an effective strategy to delay IVDD. In this study, we assessed whether umbilical cord mesenchymal stem cell-exosomes (UCMSC-exos) can be used to treat IVDD by suppressing ROS production caused by mitochondrial dysfunction. Materials and methodsHuman UCMSC-exos were isolated and identified. Nucleus pulposus cells (NPCs) were stimulated with H2O2 in the presence or absence of exosomes. Then, 4D label free quantitative (4D-LFQ) proteomics were used to analyze the differentially expressed (DE) proteins. Mitochondrial membrane potential (MMP), mitochondrial ROS and protein levels were determined via immunofluorescence staining, flow cytometry and western blotting respectively. Additionally, high-throughput sequencing was performed to identify the DE miRNAs in NPCs. Finally, therapeutic effects of UCMSC-exos were investigated in a puncture-induced IVDD rat model. Degenerative grades of rat IVDs were assessed using magnetic resonance imaging and histochemical staining. ResultsUCMSC-exos effectively improved the viability of NPCs and restored the expression of the extracellular matrix (ECM) proteins, collagen type II alpha-1 (COL2A1) and matrix metalloproteinase-13 induced by H2O2. Additionally, UCMSC-exos not only reduced the total intracellular ROS and mitochondrial superoxide levels, but also increased MMP in pathological NPCs. 4D-LFQ proteomics and western blotting further revealed that UCMSC-exos up-regulated the levels of the mitochondrial protein, mitochondrial transcription factor A (TFAM), in H2O2-induced NPCs. High-throughput sequencing and qRT-PCR uncovered that UCMSC-exos down-regulated the levels of miR-194-5p, a potential negative regulator of TFAM, induced by H2O2. Finally, in vivo results showed that UCMSC-exos injection improved the histopathological structure and enhanced the expression levels of COL2A1 and TFAM in the rat IVDD model. ConclusionsOur findings suggest that UCMSC-exos promote ECM synthesis, relieve mitochondrial oxidative stress, and attenuate mitochondrial dysfunction in vitro and in vivo, thereby effectively treating IVDD. The translational potential of this articleThis study provides solid experimental data support for the therapeutic effects of UCMSC-exos on IVDD, suggesting that UCMSC-exos will be a promising nanotherapy for IVDD.

Diagnostic and prognostic assessments of adrenocortical carcinomas by pathological features, immunohistochemical markers and reticular histochemistry staining.

Current diagnostic criteria of adrenocortical neoplasms are mostly based on morphology. The utility of immunohistochemistry (IHC) and histochemistry is limited. To evaluate the diagnostic and prognostic utility of clinicopathological features, morphology, ancillary biomarkers, and reticular histochemistry in adrenocortical neoplasms. We examined 28 adrenocortical carcinomas (ACCs) and 50 adrenocortical adenomas (ACAs) obtained from pathology archives. Clinical data were retrieved from medical records. Two pathologists independently assessed hematoxylin and eosin-stained slides, employing modified Weiss criteria for all tumors and Lin-Weiss-Bisceglia criteria for oncocytic variants. Immunohistochemical markers (Calretinin, alpha-inhibin, MelanA, SF-1, Ki-67, PHH3, IGF-2, β-catenin, P53, CYP11B1, CYP11B2, MLH1, MSH2, MSH6, PMS2, EPCAM) and Gomori's Silver histochemistry were applied. Statistical analysis utilized SPSS Statistics 26. ACCs exhibited larger tumor sizes (P<0.001) and symptomatic presentations (P = 0.031) compared to ACAs. Parameters of modified Weiss criteria and angioinvasion demonstrated diagnostic value for ACCs. Six immunohistochemical antibodies((MelanA, Ki-67, IGF-2, β-catenin, P53 and CYP11B1) and reticulin framework alterations showed diagnostic value. Notably, Ki-67 and reticulin staining were most recommended. Evident reticulin staining was frequently present in ACCs (P<0.001). Ki-67 was significantly higher in ACCs (P<0.001). Twenty-one conventional and seven oncocytic entities showed different necrosis frequencies. Symptoms and Ki-67 index ≥ 30% were prognostic for ACCs, correlating with shorter survival. This study emphasizes the diagnostic value of reticulin framework alterations and a high Ki-67 index. Markers such as CYP11B1, IGF2, P53, β-catenin and MelanA also contribute to the diagnosis of ACCs. Symptoms and Ki-67 index ≥ 30% predict shorter survival. These findings encourges the use of ancillary markers such as reticulin histochemistry and Ki-67 in the workup of evaluations of adrenocortical neoplasms.

Current state of in situ gene expression studiesin animal tissues

Morphological studies of farm animals are most often conducted using simple preparation and staining techniques. The study of the processes of embryogenesis, postembryonic features of the development of organs and tissues, as well as the effect of various substances remains to be elucidated not only using histochemical and immunohistochemical staining methods, but also using RNA in situ hybridization and transcriptome in situ sequencing. Aspects of many cellular and tissue processes for cattle, pigs and chickens in the context of comparative physiology have not yet been studied. The high productivity of farm animals is associated with the intensive functioning of all organs and systems of the body. The influence of the agricultural management of farm animals and its consequences on the development of an organism in ontogeny deserve a separate area of research from the point of in situ gene expression. Despite rapid development of transcriptome sequencing technologies, which result in the discovery of new candidate genes for many processes, RNA in situ hybridization remains the gold standard for their validation. This review briefly presents modern techniques and their modifications for studying in situ gene expression. Transcriptome studies that have been implemented in cattle, pigs and chickens as model organisms include: in situ RNA hybridization using ZZ-probes, tyramide signal amplification, hybridization chain reaction, digoxigenin-labeled probes, RT-PCR, single cell transcriptome sequencing, in situ transciptome sequencing. This paper is a review of the results of studies on cattle, pigs and chickens. The results of research in this area are relevant for understanding the features of adaptation mechanisms at the transcriptomic level in highly productive animals under industrial conditions in order to search for new markers of valuable agricultural traits. It should be noted that in literature there are very few studies using RNA in situ hybridization, despite the availability and simplicity of the method.

ZmPGIP1 regulates stem strength by enhancing lignin and cellulose biosynthesis in Arabidopsis thaliana

Polygalacturonase inhibiting proteins (PGIPs) are typical leucine-rich repeat (LRR) proteins that inhibit the activity of polygalacturonase (PG) in pathogens, but their function in promoting to plant stem development has been less studied. In this study, the PGIP gene from Zea mays was cloned and heterologously expressed in Arabidopsis thaliana, and then the plant phenotypes were observed and a set of physiological and biochemical indicators were measured. The results indicate that the ZmPGIP1 protein is expressed both in the cell membrane and cell wall. Our research observed that the heterologous expression of ZmPGIP1 significantly increased the production of lignin, cellulose and hemicellulose in transgenic lines. The stems of transgenic plants also showed a thicker cortical tissue. The increase in lignin deposition observed during histochemical staining suggests that the ZmPGIP1 gene may improve plant mechanical strength. These were tightly associated with the upregulated expression of lignin/cellulose-synthesis genes (C4H, C3H, F5H, PAL1/3, CCR1, CAD1-3/9, CCOMT, MYB58, CESA1/10) to enhance plant cell wall strength in transgenic lines. Our findings provide new perspectives on the growth regulation mechanisms of PGIP proteins in plant cell walls and provide a potential candidate gene for breeding increased disease-resistance and higher cellulose production in plants.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from − 891 bp to − 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Cloning of the Arabidopsis SMAP2 promoter and analysis of its expression activity

The SMALL ACIDIC PROTEIN (SMAP) gene is evolutionarily indispensable for organisms. There are two copies of the SMAP gene in the Arabidopsis thaliana genome, namely, SMAP1 and SMAP2. The function of SMAP2 is similar to that of SMAP1, and both can mediate 2,4-D responses in the root of Arabidopsis. This study cloned the AtSMAP2 genetic promoter sequence. Two promoter fragments of different lengths were designed according to the distribution of their cis-acting elements, and the corresponding β- glucuronidase (GUS) expression vector was constructed. The expression activity of promoters of two lengths, 1993 bp and 997 bp, was studied by the genetic transformation in Arabidopsis. The prediction results of cis-acting elements in the promoter show that there are many hormone response elements in 997 bp, such as three abscisic acid response elements ABRE, gibberellin response elements P-box and GARE-motif and auxin response element AuxRR-core. Through GUS histochemical staining and qRT‒PCR analysis, it was found that the higher promoter activity of PAtSMAP2-997, compared to PAtSMAP2-1993, drove the expression of GUS genes at higher levels in Arabidopsis, especially in the root system. The results provide an important basis for subsequent studies on the regulation of AtSMAP2 gene expression and biological functions.

Biochemical Variability of Nasal Cartilages: Implications in Functional Rhinoplasty.

Nasal valve compromise is a common issue for patients presenting to an otolaryngologist. Anatomically, the dorsal septal cartilage (DSA) articulates with the upper lateral nasal cartilage (ULC). When the ULC weakens, it is prone to collapse and may result in nasal obstruction. Our objectives were to analyze histological differences between quadrangular and DSA nasal cartilages and compare histological composition of these tissues among patients with diagnosis of nasal valve compromise (NVC) versus those without NVC (no NVC). Prospective cross-sectional study of nasal cartilages from seventy-three (73) live donors. Quadrangular cartilage (QC), and DSA from patients undergoing septorhinoplasty were collected. Safranin O histochemical staining was used to observe glycosaminoglycans (GAGs) content and cell count. Masson's Trichrome staining was used to assess collagen content in these nasal cartilages. Mean GAG content was lower in DSA compared with QUAD cartilage (68.18% vs. 87.22%, p < 0.0001), and was lowest in DSA of patients with NVC (59.07%). Mean collagen content was higher in DSA compared with QUAD cartilage, (65.43% vs. 46.65%, p = 0.0002), and was highest in DSA of patients with NVC (74.86%). The DSA showed decreased GAG and increased collagen content compared with quadrangular cartilage. Within the DSA subsite, patients with presurgical NVC had the lowest GAG and highest collagen levels. This observation suggests that the biochemical composition of the DSA may contribute to its weakening, potentially resulting in its inability to withstand minor trauma and/or the effects of aging, ultimately leading to NVC. N/A Laryngoscope, 2024.

MSC Promotes the Secretion of Exosomal lncRNA KLF3-AS1 to Regulate Sphk1 Through YY1-Musashi-1 Axis and Improve Cerebral Ischemia-Reperfusion Injury.

Stroke remains the 3rd leading cause of long-term disability globally. Over the past decade, mesenchymal stem cell (MSC) transplantation has been proven as an effective therapy for ischemic stroke. However, the mechanism of MSC-derived exosomal lncRNAs during cerebral ischemia/reperfusion (I/R) remains ambiguous. The oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) rat model were generated. MSCs were isolated and characterized by flow cytometry and histochemical staining, and MSC exosomes were purified and characterized by transmission electron microscopy, flow cytometry and Western blot. Western blot, RT-qPCR and ELISA assay were employed to examine the expression or secretion of key molecules. CCK-8 and TUNEL assays were used to assess cell viability and apoptosis. RNA immunoprecipitation and RNA pull-down were used to investigate the direct association between krüppel-like factor 3 antisense RNA 1 (KLF3-AS1) and musashi-1(MSI1). Yin Yang 1 (YY1)-mediated transcriptional regulation was assessed by chromatin immunoprecipitation and luciferase assays. The histological changes and immunoreactivity of key molecules in brain tissues were examined by H&E and immunohistochemistry. MSCs were successfully isolated and exhibited directionally differential potentials. MSC exosomal KLF3-AS1 alleviated OGD/R-induced inflammation in SK-N-SH and SH-SY5Y cells via modulating Sphk1. Mechanistical studies showed that MSI1 positively regulated KLF3-AS1 expression through its direct binding to KLF3-AS1. YY1 was identified as a transcription activator of MSI1 in MSCs. Functionally, YY1/MSI1 axis regulated the release of MSC exosomal KLF3-AS1 to modulate sphingosine kinase 1 (Sphk1)/NF-κB pathway, thereby ameliorating OGD/R- or cerebral I/R-induced injury. MSCs promote the release of exosomal KLF3-AS1 to regulate Sphk1 through YY1/MSI axis and improve cerebral I/R injury.

Detection of edible insect as a component of snack bars using histochemical method

The edible insects are commonly used in food products, but the methods for detection are not yet established. This study presents a new bright-field method for detecting insects in food using histochemical staining (PAS-Calleja and Pasini). For the purpose of the study, snack bars from house cricket (Acheta domesticus) and mealworm (Tenebrio molitor) were produced in the concentration range of 0.0–25.0%, and for the purpose of verification, also samples from the market network were obtained. A 100% consensus of evaluators on the presence of both insect species was demonstrated in the snack bars from the concentration of 2.5%. Sensitivity and specificity were 100%, reliability was 95.4% for PAS-Calleja staining For Pasini staining sensitivity was 93%, specificity was also 100% as PAS-Calleja staining and reliability was 94.2%. The cuticle appears to be crucial for evaluating the presence of insects due to its high affinity for the PAS-Calleja staining used. A homogeneity test was carried out within the evaluation. The distance between individual fragments of insects was statistically significant at low concentrations. In conclusion, it can be assessed that evaluators can confirm the positivity of a bar samples for the presence of insects (ACH and TM) from a 1% content using the PAS-C staining histochemical method.

Transcriptome analysis and functional validation reveal the novel role of LhCYCL in axillary bud development in hybrid Liriodendron.

Shoot branching significantly influences yield and timber quality in woody plants, with hybrid Liriodendron being particularly valuable due to its rapid growth. However, understanding of the mechanisms governing shoot branching in hybrid Liriodendron remains limited. In this study, we systematically examined axillary bud development using morphological and anatomical approaches and selected four distinct developmental stages for an extensive transcriptome analysis. A total of 9,449 differentially expressed genes have been identified, many of which are involved in plant hormone signal transduction pathways. Additionally, we identified several transcription factors downregulated during early axillarybud development, including a noteworthy gene annotated as CYC-like from the TCP TF family, which emerged as a strong candidate for modulating axillary bud development. Quantitative real-time polymerase chain reaction results confirmed the highest expression levels of LhCYCL in hybrid Liriodendron axillary buds, while histochemical β-glucuronidase staining suggested its potential role in Arabidopsis thaliana leaf axil development. Ectopic expression of LhCYCL in A. thaliana led to an increase of branches and a decrease of plant height, accompanied by altered expression of genes involved in the plant hormone signaling pathways. This indicates the involvement of LhCYCL in regulating shoot branching through plant hormone signaling pathways. In summary, our results emphasize the pivotal role played by LhCYCL in shoot branching, offering insights into the function of the CYC-like gene and establishing a robust foundation for further investigations into the molecular mechanisms governing axillary bud development in hybrid Liriodendron.

M2WISH: An easy and efficient protocol for whole‐mount mRNAin situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana

SUMMARYGene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co‐expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

3D inversion recovery ultrashort echo time MRI can detect demyelination in cuprizone-treated mice.

To test the ability of inversion-recovery ultrashort echo time (IR-UTE) MRI to directly detect demyelination in mice using a standard cuprizone mouse model. Non-aqueous myelin protons have ultrashort T2s and are "invisible" with conventional MRI sequences but can be detected with UTE sequences. The IR-UTE sequence uses an adiabatic inversion-recovery preparation to suppress the long T2 water signal so that the remaining signal is from the ultrashort T2 myelin component. In this study, eight 8-week-old C57BL/6 mice were fed cuprizone (n = 4) or control chow (n = 4) for 5 weeks and then imaged by 3D IR-UTE MRI. The differences in IR-UTE signal were compared in the major white matter tracts in the brain and correlated with the Luxol Fast Blue histochemical marker of myelin. IR-UTE signal decreased in cuprizone-treated mice in white matter known to be sensitive to demyelination in this model, such as the corpus callosum, but not in white matter known to be resistant to demyelination, such as the internal capsule. These findings correlated with histochemical staining of myelin content. 3D IR-UTE MRI was sensitive to cuprizone-induced demyelination in the mouse brain, and is a promising noninvasive method for measuring brain myelin content.

Increased mucous cell population and modulation of Bax/Bcl-2 factors characterize in vivo gastroprotective activity of Cissampelos owariensis in rats

The physiological integrity of the gastric mucosa is dependent on the balance between the mucosal protective and aggressive factors. Medicinal plants or their derivatives generally exhibit gastroprotective effect by promoting the protective factors against the aggressive factors of the gastric mucosa. The study was conducted to elucidate the gastroprotective mechanism of the methanol extract of C. owariensis (MECo) in rats. Twenty male Wistar rats were divided into four groups, which include control groups A and B – given distilled water – and treated groups C and D – animals given 100 and 300 mg/kg MECo respectively for 28 days. After the treatment period, gastric mucosal injury was induced for groups B-D by pyloric ligation method. The gastric tissue of animals was collected, processed for histology (haematoxylin and eosin technique), histochemistry (periodic acid-schiff technique) and immunohistochemical staining (for Bcl-2 &amp; Bax proteins). The results of the gastric histomorphology showed prominent and widespread mucosal erosion in positive control group B compared to normal control group A, while treated groups C and D showed only mild or focal mucosal erosion. Furthermore, the histochemical results showed significant increase in mucous cell population in treated groups C and D compared to positive control group B. The immunostaining results showed significant up-regulation of anti-apoptotic Bcl-2 protein and down-regulation of pro-apoptotic Bax protein in the treated groups C and D compared to the control groups A and B. In conclusion, the findings of this study indicate that the increased mucous cell population and modulation of apoptotic signaling highlights the mechanism of gastroprotective activity of MECo.

Copper hepatopathies in Australian dogs.

To evaluate hepatopathies in Australian dogs according to the World Small Animal Veterinary Association (WSAVA) guidelines. Specifically, to describe the prevalence and survival of dogs with copper-associated hepatopathy. Medical records from the Small Animal Specialist Hospital were reviewed to identify dogs with liver disease and liver biopsy between November 2008 and November 2021. Liver histopathology reports were reviewed with a board-certified veterinary pathologist and classified according to the WSAVA guidelines. Histopathology reports and clinical records were reviewed to ascertain the most important histological process for statistical analysis. Copper-associated hepatopathy was defined as (i) histological evidence of copper accumulation in centrilobular areas (Zone 3) associated with hepatocyte necrosis, inflammation with copper-laden macrophages and chronic hepatitis (ii) histochemical copper staining showing hepatocyte copper accumulation in the centrilobular areas and iii) hepatic copper measurement with concentrations greater than 600 μg/g dry weight of liver. Dogs with primary inflammatory parenchymal disease included dogs with copper-associated hepatopathy, idiopathic chronic hepatitis, non-specific reactive hepatitis, chronic bacterial hepatitis and immune-mediated chronic hepatitis. Descriptive statistics were performed for all dogs. Age, weight and clinicopathologic data were compared between dogs with copper-associated hepatopathy and dogs with other causes of chronic primary inflammatory parenchymal liver disease (Kruskal-Wallis test). Survival times were calculated and compared (Kaplan-Meier curves and log rank test) between dogs with copper-associated hepatopathy and dogs with other chronic primary inflammatory parenchymal liver diseases. Breed was evaluated to determine the breed most commonly affected with copper-associated hepatopathy and identify any breed in which this disease has not previously been described. Sixty-seven (43 female, 24 male) dogs with a median age of 7.8 years (quartile [Q] Q1-Q3 4.5-9.6 years) were included. Thirteen dogs had copper-associated hepatopathy, eight dogs had idiopathic chronic hepatitis, eight dogs had non-specific reactive hepatitis, seven dogs had disorders associated with portal hypertension, five dogs had chronic bacterial hepatitis and four dogs had immune-mediated chronic hepatitis. Compared with dogs with other causes of chronic primary inflammatory parenchymal liver disease, dogs with copper-associated hepatopathy tended to be younger (6.73 vs. 8.01 years, P = 0.057) and heavier (19.8 vs. 9.6 kg, P = 0.052) than dogs with other causes of primary chronic inflammatory parenchymal diseases. There was no statistically significant difference when ALT (P = 0.30), ALP (P = 0.18) and total bilirubin (P = 0.13) were compared between the two groups. The median survival time for all dogs after liver biopsy was 2010 days (CI 1321 days - not reached). There was no significant difference in survival between dogs with copper-associated hepatopathy and dogs with other causes of chronic primary inflammatory parenchymal liver disease (P = 0.5). Copper-associated hepatopathy was common among Australian dogs with chronic hepatopathies, occurring in younger and heavier dogs than other causes of primary inflammatory parenchymal liver disease. Clinical pathology is not useful for differentiating between copper-associated hepatopathy and other causes of chronic primary inflammatory parenchymal liver disease. When copper-associated hepatopathy is treated, the prognosis can be good. This is the first report of copper-associated hepatopathy in Australian Cavalier King Charles Spaniels.

Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

The Role of Collagen VIII in the Aging Mouse Kidney.

The gradual loss of kidney function due to increasing age is accompanied by structural changes such as fibrosis of the tissue. The underlying molecular mechanisms are complex, but not yet fully understood. Non-fibrillar collagen type VIII (COL8) could be a potential factor in the fibrosis processes of the aging kidney. A pathophysiological significance of COL8 has already been demonstrated in the context of diabetic kidney disease, with studies showing that it directly influences both the development and progression of renal fibrosis occurring. The aim of this study was to investigate whether COL8 impacts age-related micro-anatomical and functional changes in a mouse model. The kidneys of wild-type (Col8-wt) and COL8-knockout (Col8-ko) mice of different age and sex were characterized with regard to the expression of molecular fibrosis markers, the development of nephrosclerosis and renal function. The age-dependent regulation of COL8 mRNA expression in the wild-type revealed sex-dependent effects that were not observed with collagen IV (COL4). Histochemical staining and protein analysis of profibrotic cytokines TGF-β1 (transforming growth factor) and CTGF (connective tissue growth factor) in mouse kidneys showed significant age effects as well as interactions of the factors age, sex and Col8 genotype. There were also significant age and Col8 genotype effects in the renal function data analyzed by urinary cystatin C. In summary, the present study shows, for the first time, that COL8 is regulated in an age- and sex-dependent manner in the mouse kidney and that the expression of COL8 influences the severity of age-induced renal fibrosis and function.

Cloning and Functional Analysis of CsROP5 and CsROP10 Genes Involved in Cucumber Resistance to Corynespora cassiicola.

The cloning of resistance-related genes CsROP5/CsROP10 and the analysis of their mechanism of action provide a theoretical basis for the development of molecular breeding of disease-resistant cucumbers. The structure domains of two Rho-related guanosine triphosphatases from plant (ROP) genes were systematically analyzed using the bioinformatics method in cucumber plants, and the genes CsROP5 (Cucsa.322750) and CsROP10 (Cucsa.197080) were cloned. The functions of the two genes were analyzed using reverse-transcription quantitative PCR (RT-qPCR), virus-induced gene silencing (VIGS), transient overexpression, cucumber genetic transformation, and histochemical staining technology. The conserved elements of the CsROP5/CsROP10 proteins include five sequence motifs (G1-G5), a recognition site for serine/threonine kinases, and a hypervariable region (HVR). The knockdown of CsROP10 through VIGS affected the transcript levels of ABA-signaling-pathway-related genes (CsPYL, CsPP2Cs, CsSnRK2s, and CsABI5), ROS-signaling-pathway-related genes (CsRBOHD and CsRBOHF), and defense-related genes (CsPR2 and CsPR3), thereby improving cucumber resistance to Corynespora cassiicola. Meanwhile, inhibiting the expression of CsROP5 regulated the expression levels of ROS-signaling-pathway-related genes (CsRBOHD and CsRBOHF) and defense-related genes (CsPR2 and CsPR3), thereby enhancing the resistance of cucumber to C. cassiicola. Overall, CsROP5 and CsROP10 may participate in cucumber resistance to C. cassiicola through the ROS and ABA signaling pathways.

Advancing tomato crop protection: Green leaf volatile-mediated defense mechanisms against Nesidiocoris tenuis plant damage

Although Nesidiocoris tenuis is highly effective as a biological control agent, it can also damage tomato plants due to its zoophytophagous behavior. When N. tenuis pierces the stems and petioles of tomato plants with its stylets, it triggers callose deposition and subsequent cell death, resulting in blocked nutrient transport, floral abortions, or wilting of tender shoots. Recently, it has been shown that exposure of tomato plants to the green leaf volatile (Z)-3-hexenyl propanoate [(Z)-3-HP] activates defensive mechanisms, including the regulation of genes involved in the synthesis and degradation of callose. In this study, conducted under greenhouse conditions, we tested the hypothesis that damage caused by N. tenuis could be reduced by exposing tomato plants to (Z)-3-HP through polymeric dispensers. Tomato plants exposed to (Z)-3-HP and non-exposed control plants were inoculated with N. tenuis. Nesidiocoris tenuis established in both groups with no significant differences between the two treatments. However, as hypothesized, the damage caused by N. tenuis was significantly lower in the plants exposed to (Z)-3-HP. Gene expression analysis of salicylic, jasmonic, and abscisic acids, along with histochemical staining methods, was used to compare the defensive responses of tomato plants infested solely with N. tenuis versus those infested with N. tenuis and exposed to (Z)-3-HP. Our findings confirm the influence of (Z)-3-HP exposure on differential defensive activation between treatments and reduced callose deposition in (Z)-3-HP-exposed plants. These results pave the way for improved management of N. tenuis by enhancing the plant's defenses based on inter-plant communication.

Characterization of SIPs-type aquaporins and their roles in response to environmental cues in rice (Oryza sativa L.)

BackgroundAquaporins (AQPs) facilitate water diffusion across biological membranes and are involved in all phases of growth and development. Small and basic intrinsic proteins (SIPs) belong to the fourth subfamily of the plant AQPs. Although SIPs are widely present in higher plants, reports on SIPs are limited. Rice is one of the major food crops in the world, and water use is an important factor affecting rice growth and development; therefore, this study aimed to provide information relevant to the function and environmental response of the rice SIP gene family.ResultsThe rice (Oryza sativa L. japonica) genome encodes two SIP-like genes, OsSIP1 and OsSIP2, whose products are predominantly located in the endoplasmic reticulum (ER) membrane but transient localization to the plasma membrane is not excluded. Heterologous expression in a yeast aquaglyceroporin-mutant fps1Δ showed that both OsSIP1 and OsSIP2 made the cell more sensitive to KCl, sorbitol and H2O2, indicating facilitated permeation of water and hydrogen peroxide. In addition, the yeast cells expressing OsSIP2 were unable to efflux the toxic methylamine taken up by the endogenous MEP permeases, but OsSIP1 showed subtle permeability to methylamine, suggesting that OsSIP1 may have a wider conducting pore than OsSIP2. Expression profiling in different rice tissues or organs revealed that OsSIP1 was expressed in all tissues tested, whereas OsSIP2 was preferentially expressed in anthers and weakly expressed in other tissues. Consistent with this, histochemical staining of tissues expressing the promoter-β-glucuronidase fusion genes revealed their tissue-specific expression profile. In rice seedlings, both OsSIPs were upregulated to varied levels under different stress conditions, including osmotic shock, high salinity, unfavorable temperature, redox challenge and pathogen attack, as well as by hormonal treatments such as GA, ABA, MeJA, SA. However, a reduced expression of both OsSIPs was observed under dehydration treatment.ConclusionsOur results suggest that SIP-like aquaporins are not restricted to the ER membrane and are likely to be involved in unique membrane functions in substrate transport, growth and development, and environmental response.

Biochemical, morphological and molecular assessments of n butanol fraction of Phoenix dactylifera L. following exposure to inorganic mercury on the liver of Wistar rats

BackgroundMercury chloride (HgCl2) damages tissues it comes in contact with in sufficient concentration. This study evaluated the protective effects of n-butanol fraction of Phoenix dactylifera (BFPD) on mercury-triggered liver toxicity in Wistar rats. 25 male rats were divided into 5 groups of 5 rats each. Group I was administered 2 ml/kg of distilled water; group II was administered 5 mg/kg of HgCl2; group III was administered 500 mg/kg of BFPD + 5 mg/kg of HgCl2; group IV was administered 1000 mg/kg of BFPD + 5 mg/kg of HgCl2, while group V was administered 100 mg/kg of silymarin + 5 mg/kg of HgCl2. orally for 2 weeks. The rats were euthanized and liver tissue blood samples were collected for histological, histochemical, stereological, immunohistochemical, molecular, and biochemical studies.ResultsThe results revealed that HgCl2 induced oxidative stress in the rats evident by histoarchitectural distortions and altered levels of liver enzymes, proteins, and oxidative stress biomarkers when compared to the control. However, BFPD treatment restored these changes. Glutathione peroxidase levels decreased (p < 0.05) in the HgCl2−treated group when compared to the control and BFPD-treated groups. HgCl2 group revealed reduced reactivity with histochemical and immunohistochemical stains (Masson’s Trichrome and B cell Lymphoma 2) when compared to the control, with a significant decrease in quantified liver Bcl-2 stain intensity when compared to the silymarin-treated group. BFPD administration revealed normal staining intensity comparable to the control. HgCl2 administration revealed a remarked decrease in the number of hepatocytes when compared to the control, BFPD, and silymarin groups. BFPD preserved (p < 0.05) the stereological features when compared to the HgCl2-treated group. GPx activity in the liver decreased (p < 0.05) with HgCl2 administration when compared to the control and silymarin-treated groups. BFPD attenuated GPx gene activity to levels similar to the control indicating some level of amelioration against HgCl2-induced toxicity.ConclusionsThe ability of BFPD to mitigate HgCl2 triggered liver alterations could be attributed to the antioxidant property of its flavonoid content. Therefore, BFPD may be a potential candidate for treating and managing liver-induced mercury intoxication.