Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

Impact of heparanase-2 (Hpa2) on cancer and inflammation: Advances and paradigms.

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.

Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice.

BACKGROUND AND AIMS: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. METHODS: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE-/-) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. RESULTS: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE-/- mice were significantly decreased compared to control ApoE-/-mice at both disease stages. CONCLUSIONS: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular disease.

Quantitative Estimation of Promoter Activity in Cannabis sativa Using Agroinfiltration-Based Transient Gene Expression.

To properly assess promoter activity, which is critical for understanding biosynthetic pathways in different plant species, we use agroinfiltration-based transient gene expression assay. We compare the activity of several known promoters in Nicotiana benthamiana with their activity in Cannabis sativa (both hemp and medicinal cannabis), which has attracted much attention in recent years for its industrial, medicinal, and recreational properties. Here we describe an optimized protocol for transient expression in Cannabis combined with a ratiometric GUS reporter system that allows more accurate evaluation of promoter activity and reduces the effects of variable infiltration efficiency.

Nuclear localization of heparanase 2 (Hpa2) attenuates breast carcinoma growth and metastasis.

Unlike the intense research effort devoted to exploring the significance of heparanase in cancer, very little attention was given to Hpa2, a close homolog of heparanase. Here, we explored the role of Hpa2 in breast cancer. Unexpectedly, we found that patients endowed with high levels of Hpa2 exhibited a higher incidence of tumor metastasis and survived less than patients with low levels of Hpa2. Immunohistochemical examination revealed that in normal breast tissue, Hpa2 localizes primarily in the cell nucleus. In striking contrast, in breast carcinoma, Hpa2 expression is not only decreased but also loses its nuclear localization and appears diffuse in the cell cytoplasm. Importantly, breast cancer patients in which nuclear localization of Hpa2 is retained exhibited reduced lymph-node metastasis, suggesting that nuclear localization of Hpa2 plays a protective role in breast cancer progression. To examine this possibility, we engineered a gene construct that directs Hpa2 to the cell nucleus (Hpa2-Nuc). Notably, overexpression of Hpa2 in breast carcinoma cells resulted in bigger tumors, whereas targeting Hpa2 to the cell nucleus attenuated tumor growth and tumor metastasis. RNAseq analysis was performed to reveal differentially expressed genes (DEG) in Hpa2-Nuc tumors vs. control. The analysis revealed, among others, decreased expression of genes associated with the hallmark of Kras, beta-catenin, and TNF-alpha (via NFkB) signaling. Our results imply that nuclear localization of Hpa2 prominently regulates gene transcription, resulting in attenuation of breast tumorigenesis. Thus, nuclear Hpa2 may be used as a predictive parameter in personalized medicine for breast cancer patients.

Clinical manifestations and genetic mutation analysis of patients with mucopolysaccharidosis type VII in China.

OBJECTIVE: This study aimed to explore the clinical and genetic features of Chinese patients with mucopolysaccharidosis type VII (MPS VII), thereby improving early detection, disease management, and patient outcomes. METHODS: A retrospective review of medical records for five patients presenting with coarse facial features, rib protrusion, chest deformities, and scoliosis was conducted. Exome sequencing was employed to identify causative genetic mutations. RESULTS: The study comprised five patients (four males, one female) with disease onset at six months of age (range: 0-1.5 years). Common symptoms included coarse facial features, skeletal abnormalities, delayed motor and language development, and intellectual disability. Approximately 80% of the patients exhibited multiple skeletal dysplasias, enlarged adenoids or tonsils, and snoring; 60% had hernias; 40% reported hearing loss and hepatosplenomegaly. Less frequent manifestations were short stature, valvular heart disease, non-immune hydrops fetalis, and corneal opacity. All patients demonstrated elevated urine glycosaminoglycans levels and absent ß-glucuronidase activity in leukocytes. Exome sequencing identified compound heterozygous mutations in the GUSB gene in all four tested patients, uncovering seven mutations in total, three of which were novel (c.189G > A, c.869C > T, and c.1745 T > C). Furthermore, prenatal diagnosis through chorionic villus sampling in subsequent pregnancies of one patient's mother revealed both fetuses had normal ß-glucuronidase activity and no disease-causing mutations in the GUSB gene. CONCLUSION: The study's patients all presented with classic symptoms of MPS VII due to ß-glucuronidase deficiency, with three new pathogenic mutations identified in the GUSB gene. Genetic counseling and prenatal testing were highlighted as crucial for disease prevention.

Production of bilirubin via whole-cell transformation utilizing recombinant Corynebacterium glutamicum expressing a ß-glucuronidase from Staphylococcus sp. RLH1.

Bilirubin, a key active ingredient of bezoars with extensive clinical applications in China, is produced through a chemical process. However, this method suffers from inefficiency and adverse environmental impacts. To address this challenge, we present a novel and efficient approach for bilirubin production via whole-cell transformation. In this study, we employed Corynebacterium glutamicum ATCC13032 to express a ß-glucuronidase (StGUS), an enzyme from Staphylococcus sp. RLH1 that effectively hydrolyzes conjugated bilirubin to bilirubin. Following the optimization of the biotransformation conditions, a remarkable conversion rate of 79.7% in the generation of bilirubin was obtained at temperate 40 °C, pH 7.0, 1 mM Mg2+ and 6 mM antioxidant NaHSO3 after 12 h. These findings hold significant potential for establishing an industrially viable platform for large-scale bilirubin production.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue.

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Relationship of the bone phenotype of the Klotho mutant mouse model of accelerated aging to changes in skeletal architecture that occur with chronological aging.

Introduction: Due to the relatively long life span of rodent models, in order to expediate the identification of novel therapeutics of age related diseases, mouse models of accelerated aging have been developed. In this study we examined skeletal changes in the male and female Klotho mutant (kl/kl) mice and in male and female chronically aged mice to determine whether the accelerated aging bone phenotype of the kl/kl mouse reflects changes in skeletal architecture that occur with chronological aging. Methods: 2, 6 and 20-23 month old C57BL/6 mice were obtained from the National Institute of Aging aged rodent colony and wildtype and kl/kl mice were generated as previously described by M. Kuro-o. Microcomputed tomography analysis was performed ex vivo to examine trabecular and cortical parameters from the proximal metaphyseal and mid-diaphyseal areas, respectively. Serum calcium and phosphate were analyzed using a colorimetric assay. The expression of duodenal Trpv6, which codes for TRPV6, a vitamin D regulated epithelial calcium channel whose expression reflects intestinal calcium absorptive efficiency, was analyzed by quantitative real-time PCR. Results and discussion: Trabecular bone volume (BV/TV) and trabecular number decreased continuously with age in males and females. In contrast to aging mice, an increase in trabecular bone volume and trabecular number was observed in both male and female kl/kl mice. Cortical thickness decreased with advancing age and also decreased in male and female kl/kl mice. Serum calcium and phosphate levels were significantly increased in kl/kl mice but did not change with age. Aging resulted in a decline in Trpv6 expression. In the kl/kl mice duodenal Trpv6 was significantly increased. Our findings reflect differences in bone architecture as well as differences in calcium and phosphate homeostasis and expression of Trpv6 between the kl/kl mutant mouse model of accelerated aging and chronological aging. Although the Klotho deficient mouse has provided a new understanding of the regulation of mineral homeostasis and bone metabolism, our findings suggest that changes in bone architecture in the kl/kl mouse reflect in part systemic disturbances that differ from pathophysiological changes that occur with age including dysregulation of calcium homeostasis that contributes to age related bone loss.

Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a ß-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

Expression analysis of genes encoding extracellular leucine-rich repeat proteins in Arabidopsis thaliana.

Leucine-rich repeat (LRR)-containing proteins have been identified in diverse species, including plants. The diverse intracellular and extracellular LRR variants are responsible for numerous biological processes. We analyzed the expression patterns of Arabidopsis thaliana extracellular LRR (AtExLRR) genes, 10 receptor-like proteins, and 4 additional genes expressing the LRR-containing protein by a promoter: ß-glucuronidase (GUS) study. According to in silico expression studies, several AtExLRR genes were expressed in a tissue- or stage-specific and abiotic/hormone stress-responsive manner, indicating their potential participation in specific biological processes. Based on the promoter: GUS assay, AtExLRRs were expressed in different cells and organs. A quantitative real-time PCR investigation revealed that the expressions of AtExLRR3 and AtExLRR9 were distinct under various abiotic stress conditions. This study investigated the potential roles of extracellular LRR proteins in plant growth, development, and response to various abiotic stresses.

The current and emerging Klotho-enhancement strategies.

Klotho is well known as a gene with antiaging properties. It has membrane and soluble forms, providing a unique system that controls various metabolic processes essential to health and disease. Klotho deficiency has been revealed to be associated with various aging-related disorders. Based on its various known and unknown protective properties, upregulating the Klotho gene may be a possible therapeutic and/or preventive approach in aging-related complications. Some agents, such as hormonal compounds, renin-angiotensin system inhibitors, antioxidants, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, statins, vitamin D receptor agonists, antioxidants, anti-inflammatory agents, mammalian target of rapamycin (mTOR) signaling inhibitors, and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors, can possibly lead to the upregulation and elevation of Klotho levels. Demethylation and deacetylation of the Klotho gene can also be considered other possible Klotho-enhancement methods. Some emerging techniques, such as RNA modifications, gene therapy, gene editing, and exosome therapy, probably have the potential to be applied for increasing Klotho. In the present study, these current and emerging Klotho-enhancement strategies and their underlying mechanisms were comprehensively reviewed, which could highlight some potential avenues for future research.

Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach.

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42-100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5'-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.

Revealing the tumor suppressive sequence within KL1 domain of the hormone Klotho.

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1-340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1-320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/ß-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and ß sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho's tumor suppressor activity and reveals a critical role in the 320-340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.

Clearing of Vascular Tissue in Arabidopsis thaliana for Reporter Analysis of Gene Expression.

To study the gene regulatory mechanisms modulating development is essential to visualize gene expression patterns at cellular resolution. However, this kind of analysis has been limited as a consequence of the plant tissues' opacity. In the last years, ClearSee has been increasingly used to obtain high-quality imaging of plant tissue anatomy combined with the visualization of gene expression patterns. ClearSee is established as a major tissue clearing technique due to its simplicity and versatility.In this chapter, we outline an easy-to-follow ClearSee protocol to analyze gene expression of reporters using either ß-glucuronidase (GUS) or fluorescent protein (FP) tags, compatible with different dyes to stain cell walls. We detail materials, equipment, solutions, and procedures to easily implement ClearSee for the study of vascular development in Arabidopsis thaliana, but the protocol can be easily adapted to a variety of plant tissues in a wide range of plant species.

Prenatal diagnosis of mucopolysaccharidosis type VII facilitating treatment in neonatal period.

Duo exome testing was performed on a fetus conceived via in vitro fertilization with an egg donor. The fetus presented with non-immune hydrops fetalis (NIHF) at 20 + 0 weeks gestation. Two variants were detected in the GUSB gene. Biallelic pathogenic variants cause mucopolysaccharidosis type VII (MPS-VII), which can present with NIHF prenatally. At the time of analysis and initial report, one variant was classified as likely pathogenic and the other as of uncertain clinical significance. Biochemical testing of the amniotic fluid supernatant showed elevated glycosaminoglycans and low ß-glucuronidase activity consistent with the diagnosis of MPS-VII. This evidence allowed the upgrade of the pathogenicity for both variants, confirming the diagnosis of MPS-VII. The infant was born at 36 + 5 weeks and enzyme replacement therapy (ERT) using vestronidase was initiated at 20 days with planning for hematopoietic stem cell transplant ongoing. The ERT therapy has been well tolerated, with decreasing quantitative urine glycosaminoglycans. Long-term follow up is required to determine whether treatment has been successful. This case demonstrates the utility of alternative testing methods to clarify the pathogenicity of variants and the clinical utility of obtaining a diagnosis antenatally in facilitating treatment in the neonatal period, and specifically highlights MPS-VII as a treatable cause of NIHF.

Emerging role of α-Klotho in energy metabolism and cardiometabolic diseases.

BACKGROUND AND AIM: Klotho was first identified as a gene associated with aging and longevity in 1997. α-Klotho is an anti-aging protein and its role in energy metabolism, various cardiovascular diseases (CVDs), and metabolic disorders is increasingly being recognized. In this review, we aimed to outline the potential protective role and therapeutic prospects of α-Klotho in energy metabolism and cardiometabolic diseases (CMDs). METHODS: We comprehensively reviewed the relevant literature in PubMed using the keywords 'Klotho', 'metabolism', 'cardiovascular', 'diabetes', 'obesity', 'metabolic syndrome', and 'nonalcoholic fatty liver disease'. RESULTS: α-Klotho can be divided into membrane-bound Klotho, secreted Klotho, and the most studied circulating soluble Klotho that can act as a hormone. Klotho gene polymorphisms have been implicated in energy metabolism and CMDs. α-Klotho can inhibit insulin/insulin growth factor-1 signaling and its overexpression can lead to a 'healthy insulin resistance' and may exert beneficial effects on the regulation of glycolipid metabolism and central energy homeostasis. α-Klotho, mainly serum Klotho, has been revealed to be protective against CVDs, diabetes and its complications, obesity, and nonalcoholic fatty liver disease. Human recombinant Klotho protein/Klotho gene delivery, multiple drugs, or natural products, and exercise can increase α-Klotho expression. CONCLUSION: Overall, α-Klotho has demonstrated its potential as a promising target for modulating energy metabolism and CMDs, and further research is needed to explore its utilization in clinical practice in the future.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes.

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.

LncRNA 152 attenuates lipopolysaccharide-induced acute kidney injury in rats by regulating the FGF23/Klotho/MAPK axis.

AIM: This study aimed to explore the effect and related mechanisms of LncRNA 152 in acute kidney injury (AKI). METHODS: QRT-PCR was used to detect the expression of LncRNA 152, FGF23 and Klotho in the serum of patients with AKI. Subsequently, Sprague Dawley (SD) rats were induced into AKI animal model by lipopolysaccharide (LPS). Then, H&E staining was performed to observe the pathological changes in the rat kidney tissues; qRT-PCR to detect the expression of LncRNA 152, FGF23 and Klotho in the rat kidney tissues; biochemical assay and ELISA to assess the levels of renal function indexes and inflammatory factors in rat serum, as well as oxidative stress indexes in kidney tissues; and western blot to measure the protein expressions of FGF23, Klotho, p-p38 and p38 in rat kidney tissues. RESULTS: LncRNA 152 was significantly down-regulated in serum of AKI patients and kidney tissues of AKI rats. In AKI patients, LncRNA 152 was negatively correlated with FGF23 expression while positively correlated with Klotho expression. LncRNA 152 overexpression reduced the levels of blood urea nitrogen (BUN), creatinine (Cr) and cystatin C (Cys-C) and inflammatory factors in serum of AKI rats and attenuated pathological damage and oxidative stress of kidney tissues. In addition, LncRNA 152 overexpression also decreased FGF23 expression and p-p38/p38 ratio while up-regulated Klotho expression in the kidney tissues of AKI rats. CONCLUSION: LncRNA 152 attenuates oxidative stress and inflammatory responses by regulating the FGF23/Klotho axis and inhibiting the MAPK signalling pathway in rat kidney tissues, thereby ameliorating LPS-induced AKI.

Impact of Intron and Retransformation on Transgene Expression in Leaf and Fruit Tissues of Field-Grown Pear Trees.

Stable and high expression of introduced genes is a prerequisite for using transgenic trees. Transgene stacking enables combining several valuable traits, but repeated transformation increases the risk of unintended effects. This work studied the stability and intron-mediated enhancement of uidA gene expression in leaves and different anatomical parts of pear fruits during field trials over 14 years. The stability of reporter and herbicide resistance transgenes in retransformed pear plants, as well as possible unintended effects using high-throughput phenotyping tools, were also investigated. The activity of ß-glucuronidase (GUS) varied depending on the year, but silencing did not occur. The uidA gene was expressed to a maximum in seeds, slightly less in the peel and peduncles, and much less in the pulp of pear fruits. The intron in the uidA gene stably increased expression in leaves and fruits by approximately twofold. Retransformants with the bar gene showed long-term herbicide resistance and exhibited no consistent changes in leaf size and shape. The transgenic pear was used as rootstock and scion, but grafted plants showed no transport of the GUS protein through the graft in the greenhouse and field. This longest field trial of transgenic fruit trees demonstrates stable expression under varying environmental conditions, the expression-enhancing effect of intron and the absence of unintended effects in single- and double-transformed woody plants.