Real-time PCR Research Articles

Assessing the diagnostic potential of SATB2 and β-catenin as biomarkers and therapeutic targets in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis. The roles of the transcription factor special AT-rich binding protein-2 (SATB2) and β-catenin in PDAC have been a subject of controversy. We aimed to assess the diagnostic and prognostic impact of SATB2 and β-catenin in PDAC. We analyzed 44 paraffin-embedded tissue blocks along with corresponding blood and pancreatic tissues. We evaluated SATB2 expression using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). β-catenin was assessed using IHC and real-time polymerase chain reaction (qPCR). High SATB2 expression and low β-catenin expression were associated with a poor prognosis in PDAC, including advanced pathological tumor stage (pT-stage), pathological lymph node stage (pN-stage), and TNM stage. We found a positive correlation between SATB2 expression assessed by IHC and the concentration of SATB2 in both serum and tissue samples measured by ELISA. We observed a positive correlation between β-catenin expression assessed by IHC and β-catenin levels measured by qPCR. SATB2 and β-catenin could provide valuable insights into the development of pancreatic cancer, and targeting them may be beneficial for the prevention and treatment of PDAC. The levels of SATB2 in serum show promise for the diagnosis and tumor invasion of pancreatic cancer.

Spinal CRH facilitates the micturition reflex via the CRH2 receptor in rats with normal bladder and bladder outlet obstruction

Lower urinary tract symptoms (LUTS) significantly affect patient quality of life. Treatment options for bladder outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH) (a common cause of LUTS) are insufficient to relieve discomfort. As the incidence of BPH is increasing, new pharmacological targets for LUTS treatment are required. Corticotropin-releasing hormone (CRH) is a neuropeptide that controls normal micturition in rodents. Herein, we investigated the role of spinal CRH in regulating micturition in sham and BOO rats, and evaluated CRH as a therapeutic target for bladder dysfunction in BOO model Sprague–Dawley rats. Histological analysis, cystometry with intrathecal administration of CRH agonists/antagonists, western blotting, and real-time PCR assessed the role of CRH and its receptors (CRHR1 and CRHR2) in micturition in sham and BOO rats. CRH administration shortened the voiding interval, while pretreatment with antagonists against CRHR2 (but not CRHR1) suppressed CRH-induced frequent voiding. Western blotting confirmed CRHR1 expression in the dorsal root ganglia (DRG) and bladder, but not the spinal cord, of rats. Real-time PCR showed higher CRHR2 mRNA expression in the spinal cord and DRG than in the bladder in both groups. Overall, spinal CRH facilitates the micturition reflex via CRHR2, and is a promising therapeutic target for LUTS.

FoxO1 promotes high glucose-induced inflammation and cataract formation via JAK1/STAT1.

To investigate whether in diabetic cataract (DC), FoxO1 regulates high glucose (HG)-induced activation of NLRC4/IL-6 inflammatory mediators in human lens epithelial cells (SRA01/04) via the JAK1/STAT1 pathway, leading to cataract formation. Expression levels of FoxO1, inflammatory factor IL-6 and inflammatory vesicle NLRC4 were examined in SRA01/04 under high glucose (HG) stress at 25-150mM. Rat lenses were also cultured using HG medium with or without the addition of the FoxO1 inhibitor AS1842856 and the JAK1 agonist RO8191. 5.5mM glucose concentration group (NG) was used as a control. Real-time PCR, Western blots, and immunofluorescent staining evaluated the mRNA and protein levels of FoxO1, NLRC4, and IL-6. Apoptosis, cell viability, and EDU Staining were also assessed. HG stimulation induced elevated FoxO1 expression and caused NLRC4/IL-6 activation in a concentration-dependent manner. Whereas knockdown of FoxO1 inhibited the high expression of NLRC4/IL-6 inflammatory mediators in response to HG stimulation. The growth of SRA01/04 was inhibited under HG condition, and the cell proliferation ability was restored and even promoted by knocking out FoxO1. HG incubation of rat lens resulted in lens clouding and cataract formation, which was prevented by AS1842856 treatment and reversed by RO8191. FoxO1 positively regulates HG-induced SRA01/04 inflammatory activation through the JAK1/STAT1 pathway and promotes DC. This provides a feasible strategy for the treatment of diabetic cataract.

The key role of the NUDT3 gene in lung adenocarcinoma progression and its association with the manganese ion metabolism family.

Lung adenocarcinoma is one of the most common malignant tumors worldwide. Its complex molecular mechanisms and high tumor heterogeneity pose significant challenges for clinical treatment. The manganese ion metabolism family plays a crucial role in various biological processes, and the abnormal expression of the NUDT3 gene in multiple cancers has drawn considerable attention. This study aims to systematically analyze the expression characteristics of the NUDT3 gene in lung adenocarcinoma and its association with tumor progression, integrating single-cell transcriptomic analysis and experimental validation using cell lines. This study employed a comprehensive set of analytical approaches. Single-cell transcriptomic analysis of two normal and four lung adenocarcinoma samples from the GSE149655 dataset was performed using the Seurat package to identify and annotate distinct cell populations, focusing on epithelial and macrophage subtypes. Non-negative matrix factorization (NMF) and gene set variation analysis (GSVA) were applied to assess the functional enrichment and expression profiles of the manganese ion metabolism family across 14 cancers. Quantitative PCR (qPCR) was conducted to evaluate the relative mRNA expression of NUDT3 in A549 lung adenocarcinoma cell lines compared to BEAS-2B normal bronchial epithelial cell lines. GAPDH was used as the reference gene for normalization, and the relative expression levels of NUDT3 in these cell lines were analyzed to confirm bioinformatics findings. NUDT3 was found to be significantly upregulated in lung adenocarcinoma and highly correlated with tumor mutation burden (TMB) and mutation enrichment (MEs). Single-cell transcriptomic analyses demonstrated elevated NUDT3 expression in lung adenocarcinoma epithelial cells, with expression levels increasing in cells associated with advanced tumor stages. Furthermore, qPCR analysis confirmed the upregulation of NUDT3 in A549 cells compared to BEAS-2B cells, consistent with transcriptomic data. These results also highlighted significant expression differences of the manganese ion metabolism family across various cancers, including BLCA, BRCA, and COAD, with notable NUDT3 mutations enriched in these cancers. This study underscores the critical role of NUDT3 in lung adenocarcinoma progression and its potential as a therapeutic target. These findings contribute to the understanding of the manganese ion metabolism family in cancer biology and provide a foundation for precision therapies targeting NUDT3 in lung adenocarcinoma.

Phyto-mediated fabrication of silver nanoparticles from Scrophularia striata extract (AgNPs-SSE): a potential inducer of apoptosis in breast cancer cells.

Breast carcinoma stands out as the most widespread invasive cancer and the top contributor to cancer-related mortality in women. Nanoparticles have emerged as promising tools in cancer detection, diagnosis, and prevention. In this study, the antitumor and apoptotic capability of silver nanoparticles synthesized through Scrophularia striata extract (AgNPs-SSE) was investigated toward breast cancer cells. The produced AgNPs-SSE were identified using scanning electron micrograph (SEM), energy-dispersive X-ray (EDAX) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD). The cell-killing effects of AgNPs-SSE on MDA-MB231 mammary carcinoma cells were evaluated in vitro using the MTT assay over 24h. Apoptosis induction was conducted by cell cycle analysis, annexin V-FITC/PI staining, reactive oxygen species (ROS) generation, and Hoechst staining. Additionally, the gene expression of βcatenin, GSK3β, and CyclinD1 was analyzed using quantitative real-time PCR (qRT-PCR). Microscopic analysis confirmed the successful fabrication of globular AgNPs-SSE, with a mean particle dimension of 19 ± 10nm. The MTT assay revealed that IC50 value of AgNPs-SSE was 48.5µg/mL for mammary carcinoma cells and 114µg/mL for normal cells. Annexin V-FITC/PI staining specified that 85.88% of cancer cells treated with AgNPs-SSE underwent either early or late apoptosis. Treatment with AgNPs-SSE also caused a considerable rise in the subG1 cell cycle population and ROS production. Furthermore, the upregulation of βcatenin and downregulation of CyclinD1 gene expression confirmed the apoptotic mechanism. In conclusion, the findings suggest that phyto-synthesized AgNPs-SSE can restrain the expansion of breast carcinoma cells and provoke apoptosis through oxidative stress. These results highlight the potential of AgNPs-SSE as an antitumor agent against breast cancer.

First clinical cases of leishmaniosis in meerkats (Suricata suricatta) housed in wildlife parks in Madrid, Spain

BackgroundIn recent years, cases of leishmaniosis have been described in animals housed in captivity in zoos in Spain [Bennett’s wallaby (Macropus rufogriseus rufogriseus), orangutan (Pongo pygmaeus pygameus), and European otter (Lutra lutra)]. Some of these zoological parks are in endemic areas for both human and animal leishmaniosis, thus it should be very important to include this zoonosis in the differential diagnosis.MethodsThe study was carried out in two zoological parks in Madrid, Madrid Zoo and Faunia, and analyzed seven meerkats. Serological tests [rK-39 and enzyme-linked immunosorbent assay (ELISA)] and molecular tests [nested polymerase chain reaction (PCR) and real-time PCR] were performed to detect Leishmania DNA. Additionally, an entomological study was carried out in both zoological parks, with molecular tests performed on female Phlebotomus perniciosus sand flies to determine their blood meal source and detect Leishmania DNA.ResultsTwo meerkats were positive for L. infantum. A 9-year-old male from the Madrid Zoo died suddenly, showing pale mucous membranes and bilateral noninflammatory alopecia and hyperpigmentation in the lateral area of the eyes. Positive results were obtained in serology, nested PCR, and real-time PCR (blood, conjunctival and oral swabs, hair, spleen, lymph node, liver, kidney, and skin), as well as numerous amastigotes in the liver and kidney tissue samples. The other meerkat, a 12-year-old male from Faunia that is still alive, presented an alopecic lesion at the base of the tail. Positive results were obtained by nested and real-time PCR from different tissues such as blood, hair, oral, and conjunctival swabs. It was treated with oral allopurinol (25 mg/kg) and miltefosine (2 mg/kg), but the molecular diagnosis remained positive after 8 months, regarding it as a mild stage of the disease. The rest of the tested meerkats were negative. The presence of P. perniciosus phlebotomine sand flies was also detected in both zoos. Although no L. infantum DNA was detected in any of sand flies analyzed, it was determined that their food sources were rabbits and humans.ConclusionsTo our knowledge, this study describes, for the first time, the detection and infection by L. infantum in meerkats (Suricata suricatta).Graphical

Epidemiologic Investigation of Protozoa and Soil-Transmitted Helminths in Starr County, Texas.

The burden of pathogenic enteric protozoa and soil-transmitted helminths among impoverished populations living on the Texas-Mexico border is unclear. We conducted a cross-sectional study on an ongoing longitudinal cohort of 616 adults residing in Starr County, Texas. A total of 359 adults were screened for four protozoa and five soil-transmitted helminths by using real-time polymerase chain reaction. This analysis identified 48 (13.4%) participants who tested positive for Blastocystis sp., three (0.8%) who tested positive for Giardia intestinalis, and one (0.3%) who tested positive for Strongyloides stercoralis and was also coinfected with Blastocystis sp. Infection was significantly associated with age, a lack of health insurance, and living outside of a colonia. We recommend additional epidemiologic investigations to examine risk factors contributing to protozoa and soil-transmitted helminth disease transmission in border counties.

Disseminated nontuberculous mycobacteriosis due to Mycobacterium avium subsp. hominissuis infection in a reindeer from a zoo.

Bacteria in the Mycobacterium tuberculosis complex and nontuberculous mycobacteria may affect a variety of animal species under human care and pose public health risks as zoonotic pathogens. A case of sudden onset of lethargy and increased respiratory effort in a 5-y-old, intact female reindeer (Rangifer tarandus) under managed care had progressed to severe dyspnea despite aggressive treatment. The animal was euthanized due to poor prognosis. Postmortem findings included: disseminated miliary nodules in the lungs, pleura, small intestine, liver, and spleen; enlarged mesenteric lymph nodes; dilated mesenteric and serosal lymphatic vessels; and renal infarcts. Histologically, granulomatous lymphadenitis and lymphangitis with intrahistiocytic acid-fast bacilli were observed. Mycobacterium sp. DNA was detected in lung via real-time PCR. Mycobacterial culture and sequencing identified Mycobacterium avium subsp. hominissuis (MAH) within pulmonary lesions. Infection with MAH has been reported in humans and many animal species; this nontuberculous mycobacterial infection may be an emerging concern in animals under managed care. To our knowledge, MAH infection has not been reported previously in reindeer.

Reduction of Low-Density Lipoprotein Cholesterol by Mesenchymal Stem Cells in a Mouse Model of Exogenous Cushing's Syndrome.

Exogenous Cushing's syndrome, which results from prolonged glucocorticoid treatment, is associated with metabolic abnormalities. Previously, we reported the inhibitory effect of tonsil-derived mesenchymal stem cell conditioned medium (T-MSC CM) on glucocorticoid signal transduction. In this study, we investigated the therapeutic efficacy of T-MSCs in a mouse model of exogenous Cushing's syndrome. Exogenous Cushing's syndrome model mice was generated by corticosterone administration in the drinking water for 5weeks, and T-MSCs were injected intraperitoneally twice during the third week. Serum lipid profiles were measured using a chemistry analyzer. HepG2 cells were treated with dexamethasone and co-cultured with T-MSCs. Expression levels of genes involved in cholesterol metabolism were examined using real-time PCR. Low-density lipoprotein receptor (LDLR) protein levels were determined using western blotting and immunohistochemistry. Liver RNA extracted from the CORT and CORT + MSC mouse groups was used for transcriptome sequencing analysis and protein-protein interaction analysis. Weight reduction and improvements in dyslipidemia by T-MSC administration were observed only in female mice. T-MSCs reduce circulating LDL cholesterol levels by downregulating liver X receptor α (LXRα)and inducible degrader of LDLR(IDOL) expression, thereby stabilizing LDLRs in the liver. Transcriptome analysis of liver tissue revealed pathways that are regulated by T-MSCs administration. Administration of MSCs to female mice receiving chronic corticosterone treatment reduced the circulating LDL cholesterol level by downregulating the LXRα-IDOL axis in hepatocytes. These results suggest that T-MSCs may offer a novel therapeutic strategy for managing exogenous Cushing's syndrome by regulating cholesterol metabolism.

HUC-MSCs Prevent Acute High-Altitude Injury through Apoe/Pdgf-b/p-Erk1/2 Axis in Mice.

The hypobaric hypoxic atmosphere can cause adverse reactions or sickness. The purpose of this study was to explore the preventive effect and mechanism of human umbilical cord mesenchymal stem cells (hUC-MSCs) on acute pathological injury in mice exposed to high-altitude. We pretreated C57BL/6 mice with hUC-MSCs via the tail vein injection, and then the mice were subjected to hypobaric hypoxic conditions for five days. The effects of hUC-MSCs on the pathological injury of lung, heart, brain were assessed by biochemical analysis, histopathological testing, quantitative real-time polymerase chain reaction (qPCR), and western blot (WB). Further, transcriptome sequencing was used to screen for the potential therapeutic targets of hUC-MSCs in acute pathological injury, the identified signaling axis was characterized using Apoe-/- mice, qPCR and WB. hUC-MSCs administration notably prevented and relieved gastrointestinal symptoms and inflammation of lung and heart, increased blood oxygen saturation and serum superoxide dismutase (SOD) level, decreased serum malondialdehyde (MDA) level, rescued lung tissue injury and myocardial mitochondrial disorder, elevated nissl bodies number in brain tissue and reduced the degree of pulmonary and cerebral edema. Furthermore, hUC-MSCs pretreatment reversed the down-regulated Apoe and up-regulated Pdgf-b and p-Erk1/2 in the lung of hypobaric hypoxic mice. Thus, hUC-MSCs protected against acute pathological injury caused by hypobaric hypoxic condition via the Apoe/Pdgf-b/p-Erk1/2 axis, and the identified pathway was confirmed by the negative results of Apoe-/- mice. hUC-MSCs possess the preventive effect on acute pathological injury caused by hypobaric hypoxia environment at high-altitude.

Lidocaine Inhibits the Proliferation of Non-Small Cell Lung Cancer and Exerts Anti-Inflammatory Effects Through the TLR-9/MyD88/NF-κB Pathway.

Lung cancer represents a significant global health burden, with non-small cell lung cancer (NSCLC) being the most common subtype. The current standard of care for NSCLC has limited efficacy, highlighting the necessity for innovative treatment options. Lidocaine, traditionally recognized as a local anesthetic, has emerged as a compound with potential antitumor and anti-inflammatory capabilities. This study was designed to explore the impact of lidocaine on NSCLC cell proliferation and inflammation, particularly focusing on the Toll-like receptor 9 (TLR)-9/MyD88/NF-κB signaling pathway. A nude mice model of NSCLC was employed, with animals receiving lidocaine at different concentrations. In vitro experiments on A549 cells involved exposure to lidocaine, followed by assessment of cell viability, cytokine expression, and TLR-9 levels using the 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, enzyme-linked immunosorbent assay, and Quantitative Real-time polymerase chain reaction (qPCR). Protein levels were evaluated via Western blot analysis. Additionally, A549 cells were transfected with a TLR-9-overexpressing lentivirus to dissect the role of TLR-9 in lidocaine's mechanism of action. Treatment with lidocaine led to a significant reduction in tumor dimensions and a decrease in inflammatory marker expression in the NSCLC mouse model. In cellular assays, lidocaine effectively suppressed A549 cell proliferation and the expression of inflammatory cytokines. The overexpression of TLR-9 partially negated the suppressive effects of lidocaine, underscoring the significance of the TLR-9/MyD88/NF-κB pathway in mediating lidocaine's effects. Lidocaine's inhibitory effects on NSCLC cell proliferation and its anti-inflammatory mechanisms are mediated through the TLR-9/MyD88/NF-κB pathway. The study's results offer promising insights into the therapeutic potential of lidocaine in NSCLC and pave the way for future investigations into its application in cancer therapy.

The effect of low energy LED red light on osteogenetic differentiation of periodontal ligament stem cell via the ERK5 signal pathway.

The purpose of this study was to examine how low-energy LED red light influences the early to middle stage of osteogenic differentiation of periodontal ligament stem cells (PDLSCs) via the ERK5 signaling pathway. METHODS: PDLSCs were extracted from periodontal membrane tissue using enzymatic digestion. At three time points of 7, 10, and 14 days after irradiation with 5J/cm2 LED red light, the expression levels of early to middle-stage osteogenic-related genes ALP, Col-1, BSP, and OPN were detected by real-time fluorescence quantitative PCR(qRT-PCR) in both control and osteogenesis experimental groups. The addition of BIX02189 could block the ERK5 signaling pathway. Under irradiation with 5J/cm2 LED red light, the expression levels of the ERK5 gene, related proteins ERK5, p-ERK5, as well as early to middle-stage osteogenic-related genes ALP, Col-1, BSP, and OPN were detected by qRT-PCR and Western blot in the osteogenic medium group and the osteogenic medium + BIX02189 group. RESULTS: Both low-energy LED red light and osteogenic medium could induce osteogenesis and differentiation of PDLSCs, upregulating the expression of ALP, Col-1, BSP, and OPN genes in PDLSCs. Their combination also produced a synergistic effect. Moreover, the ERK5 signaling pathway participated in the promoting effect of LED red light on the early to middle-stage osteogenic differentiation of PDLSCs, indicating a positive role of LED red light in this process. CONCLUSIONS: The ERK5 signaling pathway can mediate the promotion of early to middle-stage osteogenic differentiation of PDLSCs by low-energy LED red light.

Screening of Oxidative Stress-related Biomarkers and Antioxidant Drugs for Polycystic Ovary Syndrome Based on Bioinformatics and Molecular Docking.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder affecting women of reproductive age. Oxidative stress (OS) is suggested to play a significant role in the development of PCOS. Using antioxidants to reduce OS and maintain a healthy balance in the body could be a novel treatment approach for PCOS. This study analyzed transcriptome data from the Gene Expression Omnibus database, focusing on genes associated with OS. By implementing two machine learning algorithms, three OS-related biomarkers-HMOX1, MMP9, and KLF2-were successfully identified. To evaluate the diagnostic potential of these biomarkers, a Logistic regression model was employed. Additionally, granulosa cells were collected from healthy individuals and infertile women with PCOS, and the reliability of HMOX1, MMP9, and KLF2 was verified by quantitative real-time PCR experiments. Furthermore, small molecule drugs targeting proteins encoded by genes HMOX1 and MMP9 were predicted through the Drug Signature Database. Molecular docking of drugs to proteins identified two antioxidants, butein and demethoxycurcumin, as potential candidates for PCOS therapy.

Investigation of the frequency and selected prevalence factors of equid alphaherpesvirus 4 viremia in horses with acute onset of fever and respiratory signs.

Equid alphaherpesvirus 4 (EqAHV4; Orthoherpesviridae, Varicellovirus equidalpha4; equine rhinopneumonitis virus) has seldom been associated with complications such as abortion and myeloencephalopathy, given the low tendency of this virus to induce viremia. We investigated the frequency of EqAHV4 viremia in horses with fever and respiratory signs. Case selection included all equids with EqAHV4 quantitative real-time PCR (qPCR)-positive nasal secretions (defined as EqAHV4 qPCR-positive cases) submitted to a diagnostic laboratory. Controls consisted of each case submitted before and after each EqAHV4 qPCR-positive case. Purified nucleic acid from blood samples collected from EqAHV4 qPCR-positive horses and control cases was tested for EqAHV4 by qPCR. We selected 183 EqAHV4 qPCR-positive horses and 376 EqAHV4 qPCR-negative horses. In general, EqAHV4 qPCR-positive horses were younger, and had a lower rate of anorexia and a higher rate of nasal discharge compared to the EqAHV4 qPCR-negative horses. A total of 25 of 183 (13.7%) horses with EqAHV4 qPCR-positive nasal secretions tested qPCR-positive for EqAHV4 in blood. EqAHV4 viremic horses were significantly younger (p < 0.015 for group <1-y-old) and had a significantly higher occurrence of distal limb edema (p < 0.05) than EqAHV4 non-viremic horses. Our data support the observation that EqAHV4 viremia is rarely detected in EqAHV4-infected horses, which explains the low level of reported complications, such as abortion and myeloencephalopathy.

Effects of omega-3 polyunsaturated fatty acids on inflammation resolution and angiogenesis in fat grafts in a controlled mouse model.

The upregulation or delay of acute inflammation at any stage limits fat graft survival. Active endogenous inflammation resolution mechanisms and mediators are novel therapeutic tools for inflammation. This study explored the effects of supplementation of omega-3 polyunsaturated fatty acids (PUFAs) deriving specialized proresolving mediators (SPMs) on postoperative inflammation and graft survival in vivo. Fish oil (FO) (or saline for the control group) was intragastrically administered in the C57BL/6N mouse fat graft model for a week before and after transplantation. The mice were euthanized at 3, 7, 14, 30, or 90 days posttransplantation. Serum C-reactive protein (CRP) concentration was determined via enzyme-linked immunosorbent assay (ELISA); gene expression levels of inflammatory factors, perilipin-1 and vascular endothelial growth factor (VEGF) in the grafts were analyzed via quantitative real-time PCR (QPCR); hematoxylin and eosin (H&E), masson's trichrome, immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed. Omega-3 PUFAs reduced the serum CRP concentration. Additionally, in the grafts of FO group, proinflammatory factors expression was reduced while anti-inflammatory factors expression was increased; the CD11b+ IF intensity at Days 14 and 30 was reduced, the F4/80+/CD11b+ ratio at Days 3 and 7 and the CD206+/F4/80+ ratio at Days 7, 14, and 30 were increased, consistent with the results of IHC control staining (CD11b, F4/80 and CD206); the gene expression of VEGF at Day 14 and perilipin-1 at Days 30 and 90 were increased; the perilipin-1+ %area and CD31+ %area at Day 90 were increased; inflammatory cell infiltration and fibrosis were decreased. Omega-3 PUFAs can enhance inflammation resolution and angiogenesis and promotes fat graft survival in a controlled mouse model.

An Aligned-to-Random PLGA/Col1-PLGA/nHA Bilayer Electrospun Nanofiber Membrane Enhances Tendon-to-Bone Healing in a Murine Model.

The challenge of achieving effective tendon-to-bone healing remains a significant concern in sports medicine, necessitating further exploration. Biomimetic electrospun nanomaterials present promising avenues for improving this critical healing process. To investigate the biological efficacy of a novel aligned-to-random PLGA/Col1-PLGA/nHA bilayer electrospun nanofiber membrane in facilitating tendon-to-bone healing. Controlled laboratory study. The bilayer membrane's composition, combining PLGA/Col1 for tendon attachment and PLGA/nHA for bone integration, was examined using scanning electron microscopy, Fourier transform infrared spectroscopy, and mechanical testing. Positioned between the Achilles tendon and bone, its design aimed for harmonious integration with both types of tissue. In vitro, biocompatibility, cell adhesion, and proliferation of the biomaterial were evaluated using live/dead staining and the CCK-8 assay. Collagen secretion and mineralization were measured for 2 cell types. In vivo, tendon-to-bone insertion samples harvested from mice were analyzed: micro-computed tomography assessed bone formation; histological staining evaluated chondrogenesis, tendinogenesis, and the 4-layer structure of the insertion; and biomechanical testing measured insertion strength. Real-time polymerase chain reaction identified genes involved in tendon-to-bone healing, and transcriptome analysis elucidated the underlying cellular and molecular mechanisms. The optimal composition was determined as 10% 3:1 for aligned PLGA/Col1 and 9% 5:1 for PLGA/nHA. Coculture showed minimal cell death, firm cell adherence, and steady proliferation, with PLGA/Col1 enhancing collagen secretion. In vivo, the material promoted bone and cartilage formation and improved tendon-to-bone interface strength. Transcriptome analysis indicated links to TNF and NF-κB pathways and to genes IL-1β, ADAM8, and EGR2. The novel aligned-to-random PLGA/Col1-PLGA/nHA bilayer nanofiber membrane outperformed other materials in both in vitro and in vivo evaluations, significantly enhancing tendon-to-bone healing. It notably improved cartilage and bone formation, tendon maturation, and biomechanical strength at the surgical interface. These effects may be associated with the TNF and NF-κB pathways and with the genes IL-1β, ADAM8, and EGR2. This study introduces a biomimetic nanofiber membrane enhancing tendon-to-bone healing, which is crucial for sports medicine. Its efficacy in improving healing outcomes, including bone and cartilage formation and biomechanical strength, could significantly lower failure rates in surgical procedures such as rotator cuff repair and anterior cruciate ligament reconstruction. This advancement offers promising implications for patient recovery and the effectiveness of surgical interventions in tendon-to-bone injuries.

Effect of transgene on salt tolerance of tobacco.

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

Suitability of different cytological preparations for molecular analysis of advanced NSCLC.

Around 85% of non-small cell lung cancers (NSCLCs) are diagnosed at an advanced stage (IIIB to IV), where therapeutic options depend on molecular analysis. However, diagnostic material for molecular testing is often represented by cytological samples which are generally scarce and span a wide range of preparation types. Thus, the primary objective is to efficiently manage materials for molecular profiling. This study aims to evaluate the suitability of different cytological samples to assess morphological and molecular characteristics of advanced NSCLC. Sixty-seven cytological samples obtained from patients with advanced NSCLC were utilized. The series encompassed different procedure types (fine-needle aspiration cytology, transbronchial needle aspiration, effusions) processed by cell blocks in 54% (n=36), direct smears in 33% (n=22), and Liquid Based Cytology (LBC) in 13% (n=9). Cytological diagnoses were routinely performed and molecular analysis were conducted using NGS and RT-PCR methods. Adequate quantity and quality of nucleic acids were obtained from all the samples, allowing molecular profiling. Combined Next Generation Sequencing (NGS) and Real Time-Polymerase Chain Reaction (RT-PCR) analysis showed wild-type profiles in 62.7% (n=42) and mutated profiles in 37.3% (n=25) of the samples. Kirsten Rat Sarcoma Virus (KRAS) mutations were identified in 19.5% (n=13) of samples, Epidermal Growth Factor Receptor (EGFR) mutations in 10.4% (n=7) and v-raf murine sarcoma viral oncogene homolog B (BRAF) mutations in 2.9% (n=2). Identified chromosomal alterations were v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) duplication in 2,9% (n=2). The cytological sample types examined in this study proved to be suitable for molecular testing, in addition to conventional morphologic diagnosis, showing versatility and adaptability to different clinical contexts. Molecular testing on cytological samples is accurate and fast, representing a valid tool for molecular profiling of advanced NSCLC.

Absence of Functional Autoantibodies Targeting Angiotensin II Receptor Type 1 and Endothelin-1 Type A Receptor in Circulation and Purified IgG From Patients With Systemic Sclerosis.

Systemic sclerosis (SSc) is a rare but severe autoimmune disease characterized by immune dysregulation, fibrosis, and vasculopathy. Although previous studies have highlighted the presence of functional autoantibodies targeting the angiotensin II receptor type 1 (AT1) and endothelin-1 type A receptor (ETAR), leading to autoantibody-mediated receptor stimulation and subsequent activation of endothelial cells (ECs), a comprehensive understanding of the direct interaction between these autoantibodies and their receptors is currently lacking. Moreover, existing data confirming the presence of these autoantibodies in SSc often rely on similar methodologies and assays. Our aim was to replicate previous findings and to investigate the functional effects of IgG derived from patients with SSc (SSc IgG) on AT1 and ETAR signaling, the downstream EC response, and the presence of AT1-binding autoantibodies in circulation. Quantitative polymerase chain reaction and cytokine enzyme-linked immunosorbent assay, alongside a real-time cell analyzer, were used to assess receptor-specific functional characteristics of purified SSc IgG (n = 18). Additionally, a novel protein capture assay using solubilized epitope-tagged AT1 was developed to detect AT1-binding autoantibodies in plasma samples from patients with SSc (n = 28) and healthy donors (n = 14). No evidence for EC activation in an AT1- or ETAR-dependent manner was revealed. Furthermore, stimulation with SSc IgG did not induce receptor activation or alter G protein-coupled receptor signaling on agonist stimulation in a model with receptor overexpression. Lastly, no AT1-binding autoantibodies were detected in plasma samples from patients with SSc when using epitope-tagged solubilized AT1. Overall, our study did not provide evidence to support the presence of AT1- or ETAR-activating autoantibodies in purified SSc IgG or AT1-binding autoantibodies in the circulation of patients with SSc.

Single-cell analyses of intestinal epithelium reveal the dysregulation of gut immune microenvironment in systemic lupus erythematosus

BackgroundThe small intestine harbors a rich array of intestinal intraepithelial lymphocytes (IELs) that interact with structural cells to collectively sustain gut immune homeostasis. Dysregulation of gut immune homeostasis was implicated in the pathogenesis of multiple autoimmune diseases, however, whether this homeostasis is disrupted in a lupus autoimmune background remains unclear.MethodsWe performed single-cell RNA sequencing (scRNA-seq) analyses to elucidate immune and structural milieu in the intestinal epithelium of MRL/Lpr lupus mice (Lpr mice) and MRL/Mpj control mice (Mpj mice). Comprehensive analyses including unsupervised clustering, trajectories, and cellular communication were performed. The primary findings from scRNA-seq were further validated by quantitative polymerase chain reaction (qPCR), flow cytometry, and in vivo experiments including selenium supplementation.ResultsWe observed a significant reduction in CD8αα + IELs, accompanied by a marked increase in CD8αβ + IELs in Lpr mice. Additionally, subsets of CD8 + IELs exhibiting significantly enhanced effector functions were found to be markedly enriched in Lpr mice. Intercellular communication patterns within intestinal epithelial immune and structural cells were found to be specifically altered in Lpr mice. Moreover, scRNA-seq revealed significantly decreased intestinal TCRγδ T cells (γδT) associated with reduced aryl-hydrocarbon receptor repressor (AHRR) expression and subsequent oxidative stress and ferroptosis in Lpr mice. Antioxidant selenium effectively reversed the loss of γδT in Lpr mice, improved the gut barrier, and alleviated lupus symptoms.ConclusionsOur high-resolution single-cell atlas enhances the understanding of the immune and structural milieu of intestinal epithelium in lupus and provides new insights into lupus pathogenesis mediated by intestinal immune dysregulation.