Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

Effect of M0 macrophage-derived exosome miR-181d-5p targeting BCL-2 to regulate NLRP3/caspase-1/GSDMD pathway on human renal mesangial cells pyroptosis.

BACKGROUND: Lupus nephritis (LN) is a type of autoimmune disease that impacts the kidneys. Exosomes are valuable for in-depth studies of the pathogenesis of LN. This study aimed to explore miR-181d-5p expression levels in M0 macrophage-derived exosomes and their role in human renal mesangial cells (HRMC) pyroptosis through binding to BCL-2. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from patients with lupus nephritis (LN) and healthy subjects. Monocytes isolated from these samples were induced into M0 macrophages using recombinant human granulocyte colony-stimulating factor (rhG-CSF). In a parallel process, THP-1 cells were induced into M0 macrophages using Phorbol Myristate Acetate (PMA). LPS- and ATP-stimulated HRMC were used to construct a cell pyroptosis model. We then introduced different miR-181d-5p mimic fragments into the M0 macrophages derived from the THP-1 cells. Subsequently, exosomes from these macrophages were co-cultured with HRMC. To evaluate the impact on HRMC, we conducted proliferation and apoptosis assessments using CellCountingKit-8assay and flow cytometry. The effect of exosomal miR-181d-5p on HRMC pyroptosis was assessed using western blot. The miR-181d-5p and BCL-2 targeting relationship was detected using real-time fluorescence quantitative PCR. IL-6, IL-1ß, and TNF-α levels in cell supernatants were detected using ELISA kits. RESULTS: In this study, we observed an increase in miR-181d-5p levels within exosomes secreted from M0 macrophages obtained by induction of monocytes from LN patients. It was found that miR-181d-5p can target binding to BCL-2. Exosomes with elevated levels of miR-181d-5p contributed to a significant increase in miR-181d-5p within HRMC, facilitating its proliferation and inhibiting apoptosis. Furthermore, exosomes expressing high levels of miR-181d-5p were observed to promote an inflammatory response and pyroptosis in HRMC. Notably, these effects were reversed when the levels of miR-181d-5p in the exosomes were reduced. CONCLUSION: Inhibition of miR-181d-5p, derived from M0 macrophage exosomes, effectively suppresses inflammation and pyroptosis in HRMC. This discovery indicates that miR-181d-5p holds the potential as a valuable target in the development of treatments for Lupus Nephritis (LN).

MicroRNA miR-181d-5p regulates the MAPK signaling pathway by targeting mitogen-activated protein kinase 8 (MAPK8) to improve lupus nephritis.

BACKGROUND: Lupus nephritis (LN) is a common immune disease. The microRNA (miR)-181d-5p is a potential target for treating kidney injury. However, the therapeutic role of miR-181d-5p in LN has not been investigated. This study aimed to investigate the role of miR-181d-5p in targeting mitogen-activated protein kinase 8 (MAPK8) and stimulating the MAPK signaling pathway in LN. METHODS: RT-qPCR was performed to identify the variations in miR-181d-5p expression in peripheral blood mononuclear cells (PBMCs) obtained from 42 LN patients, 30 healthy individuals, 6 MRL/lpr mice and 6 C57BL/6 mice. Western blot was used to detect the effect of miR-181d-5p on the MAPK signaling pathway in THP-1 cells and MRL/lpr mice. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect the effect of miR-181d-5p on antinuclear antibodies and inflammatory factors. A dual-luciferase reporter assay was used to verify whether miR-181d-5p directly targets MAPK8. Flow cytometry was performed to evaluate apoptosis rates in transfected THP-1 cells. RESULTS: miR-181d-5p expression was downregulated in PBMCs of LN patients (P < 0.01) and MRL/lpr mice (P < 0.05). A dual luciferase reporter assay demonstrated that miR-181d-5p inhibits MAPK8 (P < 0.01). Overexpression of miR-181d-5p inhibited the phosphorylation of p38 (P < 0.001) and p44/42 (P < 0.01). Moreover, miR-181d-5p decreased the apoptosis rate of THP-1 cells (P < 0.001), and reduced the secretion of IL-6 (P < 0.01) and TNF-α (P < 0.01). Furthermore, overexpression of miR-181d-5p decreased anti-dsDNA antibody (P < 0.05), anti-Sm antibody (P < 0.01), and fibrosis levels in MRL/lpr mice. CONCLUSION: Upregulation of miR-181d-5p showed anti-inflammatory and anti-apoptotic effects on THP-1 cells in vitro and kidney injury in vivo. These effects were achieved by miR-181d-5p targeting MAPK8 to inhibit phosphorylation of p38 and p44/42. These results may offer new insights for improving therapeutic strategies against lupus nephritis.

Reactive Oxygen Species-Mediated Mitophagy and Cell Apoptosis are Involved in the Toxicity of Aluminum Chloride Exposure in GC-2spd.

Aluminum chloride is an inorganic polymeric coagulant commonly found in daily life and various materials. Although male reproductive toxicity has been associated with AlCl3 exposure, the underlying mechanism remains unclear. This study aimed to examine the impact of AlCl3 exposure on mitophagy and mitochondrial apoptosis in testicular tissue and mouse spermatocytes. Reactive oxygen species (ROS) and ATP levels were measured in GC-2spd after AlCl3 exposure using a multifunctional enzyme labeler. The changes in mitochondrial membrane potential (MMP) and TUNEL were observed through confocal laser microscopy, and the expression of proteins associated with mitophagy and apoptosis was analyzed using Western blot. Our results demonstrated that AlCl3 exposure disrupted mitophagy and increased apoptosis-related protein expression in testicular tissues. In the in vitro experiments, AlCl3 exposure induced ROS production, suppressed cell viability and ATP production, and caused a decrease in MMP, leading to mitophagy and cell apoptosis in GC-2spd cells. Intervention with N-acetylcysteine (NAC) reduced ROS production and partially restored mitochondrial function, thereby reversing the resulting mitophagy and cell apoptosis. Our findings provide evidence that ROS-mediated mitophagy and cell apoptosis play a crucial role in the toxicity of AlCl3 exposure in GC-2spd. These results contribute to the understanding of male reproductive toxicity caused by AlCl3 exposure and offer a foundation for future research in this area.

Study on reservoir optimal operation based on coupled adaptive ε constraint and multi strategy improved Pelican algorithm.

The optimal operation of reservoir groups is a strongly constrained, multi-stage, and high-dimensional optimization problem. In response to this issue, this article couples the standard Pelican optimization algorithm with adaptive ε constraint methods, and further improves the optimization performance of the algorithm by initializing the population with a good point set, reverse differential evolution, and optimal individual t-distribution perturbation strategy. Based on this, an improved Pelican algorithm coupled with adaptive ε constraint method is proposed (ε-IPOA). The performance of the algorithm was tested through 24 constraint testing functions to find the optimal ability and solve constraint optimization problems. The results showed that the algorithm has strong optimization ability and stable performance. In this paper, we select Sanmenxia and Xiaolangdi reservoirs as the research objects, establish the maximum peak-cutting model of terrace reservoirs, apply the ε-IPOA algorithm to solve the model, and compare it with the ε-POA (Pelican algorithm coupled with adaptive ε constraint method) and ε-DE (Differential Evolution Algorithm) algorithms, the results indicate that ε. The peak flow rate of the Huayuankou control point solved by the IPOA algorithm is 12,319 m3/s, which is much lower than the safe overflow flow rate of 22,000 m3/s at the Huayuankou control point, with a peak shaving rate of 44%, and other algorithms do not find effective solutions meeting the constraint conditions. This paper provides a new idea for solving the problem of flood control optimal operation of cascade reservoirs.

Maximizing the performance of protein-based fluorescent biosensors.

Fluorescent protein (FP)-based biosensors are genetically encoded tools that enable the imaging of biological processes in the context of cells, tissues, or live animals. Though widely used in biological research, practically all existing biosensors are far from ideal in terms of their performance, properties, and applicability for multiplexed imaging. These limitations have inspired researchers to explore an increasing number of innovative and creative ways to improve and maximize biosensor performance. Such strategies include new molecular biology methods to develop promising biosensor prototypes, high throughput microfluidics-based directed evolution screening strategies, and improved ways to perform multiplexed imaging. Yet another approach is to effectively replace components of biosensors with self-labeling proteins, such as HaloTag, that enable the biocompatible incorporation of synthetic fluorophores or other ligands in cells or tissues. This mini-review will summarize and highlight recent innovations and strategies for enhancing the performance of FP-based biosensors for multiplexed imaging to advance the frontiers of research.

Downregulation of the adenosine a2b receptor by RNA interference inhibits hepatocellular carcinoma cell growth.

To investigate the biological effect of adenosine A2b receptor (A2bR) on the human hepatocellular carcinoma cell line HepG2, three A2bR siRNA constructs were transiently transfected into HepG2 cells. The results showed that A2bR siRNA reduced the levels of A2bR mRNA and protein. In order to further detect the function of A2bR, we established a stable hepatocellular carcinoma cell line (HepG2) expressing siRNA targeting the adenosine A2b receptor. Targeted RNAi significantly inhibited tumor cell growth in vitro, and flow cytometry (FCM) showed that significantly more cells expressing A2bR siRNA were in the G0/G1 phase compared to the untransfected group ((89.56% ± 3.15%) versus (56.19% ± 1.58%), P < 0.01). These results indicated that silencing the expression of adenosine A2b receptor in HepG2 cells can suppress cell growth effectively by blocking the cell cycle. Downregulation of adenosine A2b receptor gene expression with RNA interference could be a new approach to hepatocellular carcinoma therapy.

[Radiofrequency ablation therapy combined with suture and ligation surgery for patients with giant cavernous hemangiomas of the liver].

OBJECTIVE: To evaluate the feasibility and efficacy of radiofrequency ablation (RFA) therapy combined with suture and ligation surgery for patients with giant hepatic cavernous hemangioma (HCH). METHODS: Between June 2004 and June 2005, a total of 30 patients were treated by RFA therapy after suture and ligation surgery (SL group, n = 15, with 18 liver lesions) or RFA therapy without suture and ligation surgery (non-SL group, n = 15, with 17 liver lesions) under general anesthesia. All patients had obvious symptoms such as abdominal discomfort, pain and swelling. Preoperative diagnosis of HCH was established by means of ultrasonography, helical computed tomography (CT) scans, and magnetic resonance imaging (MRI). The mean maximum diameter of the lesions was 8.8 cm +/- 1.4 cm. All of the 35 lesions were located on the liver surface, in the caudate lobe of the liver, or adjacent to the gallbladder. Seven patients had chronic calculous cholecystitis, 6 common duct stones, 5 thrombocytopenias, and one posthepatitic cirrhosis. Thirteen of the 30 patients had previous laparotomy. Therapeutic efficacy and clinical data of RFA therapy were compared between these two groups. RESULTS: RFA therapy under ultrasound guidance was performed successfully in all the patients. Cholecystectomy was performed simultaneously for gallstones in 7 patients and for abutting of gallbladder from hemangioma in 2 patients. Choledochotomy with T-tube drainage was performed in 6 patients. The mean blood loss, the mean RFA time per lesion and reduction rate of maximum diameter of the lesions 6 months after RFA in the SL group and non-SL group were 88.0 ml +/- 22.4 ml vs. 255.0 ml +/- 71.7 ml (P < 0.001), 23.0 min +/- 7.5 min vs. 53.3 min +/- 16.0 min (P < 0.001), and 61.8% vs. 44.8% (P < 0.001) respectively. No severe complication related to RFA was observed in all patients. At a median follow-up of 12 months (6 approximately 17 months), a complete lesion necrosis was achieved on the contrast-enhanced helical CT scans in both groups. During the follow-up, all of the 15 patients were free of upper abdominal pain in the SL group, and 12 patients were symptom-free and 3 obtained significant amelioration of symptoms in the non-SL group. CONCLUSION: RFA therapy combined with suture and ligation surgery is a feasible, safe, and effective treatment modality for patients with giant HCHs. It can reduce blood loss, shorten RFA therapy time, and increase therapeutic efficacy of RFA. Intraoperative ultrasonography is a useful adjunct for increasing the therapeutic efficacy of RFA and reducing the complications related to RFA.

[Clinical evaluation of radiofrequency ablation therapy in patients with hepatic cavernous hemangiomas].

OBJECTIVE: To evaluate the feasibility, safety and efficacy of radiofrequency ablation (RFA) therapy in patients with hepatic cavernous hemangioma (HCH) and investigate its optimal operative approach. METHODS: Between March 2001 and June 2004, a total of 68 patients, 18 males and 50 females, age 43.1 (30-64), with 104 HCHs 2.5-11 cm in diameter with the mean size of 5.6 cm, were treated by ultrasound-guided RFA, via percutaneous (n = 19), laparoscopic (n = 29), or open surgical (n = 20) approach. In 7 patients with hepatic lesions larger than 7 cm in diameter, Pringle maneuver was used to occlude the hepatic blood flow during the laparoscopic and open RFA therapy. All patients were followed up with helical computed tomographic (CT) scans and ultrasonography for 19 months (6-36 months). RESULTS: Additional intrahepatic lesions not detected preoperatively were found in 2 patients (with 2 new lesions) via laparoscopy and 3 patients (with 4 new lesions) via celiotomy. All patients were treated with RFA successfully. The mean blood loss in the Pringle group (90.0 ml +/- 22.4 ml) was significantly fewer than that in the non-Pringle group (249 ml +/- 56 ml) (P < 0.01). The mean RFA time per lesion in the Pringle group (29.0 min +/- 7.5 min) was shorter markedly compared to the non-Pringle group (55.4 min +/- 12.4 min) (P < 0.01). In the laparoscopic RFA group, laparoscopic cholecystectomy was performed simultaneously in 15 patients with chronic calculous cholecystitis and in another 2 patients because of tumors abutting the gallbladders, and laparoscopic fenestration with intraperitoneal drainage was performed in 3 patients with simple hepatic cysts. In the open RFA group, cholecystectomy was performed in 5 patients with gallbladder diseases, partial cystectomy was performed in one patient with a hepatic cyst, and choledochotomy was performed in 3 patients with common bile duct stones. Postoperative fever and abnormal serum transaminase (ALT and AST) levels were observed in 29 patients (42.6%). A transient hematuria occurred in one patient after open RFA. No specific complications developed during or after RFA. The follow-up showed a complete lesion necrosis rate of 99% (103/104). One patient showed an incomplete lesion necrosis in the margin of RFA site 6 months after percutaneous RFA therapy and obtained retreatment with percutaneous RFA. CONCLUSION: RFA therapy is a safe, feasible and effective treatment options for patients with HCHs. This procedure can be performed via percutaneous, laparoscopic, or open approach. To prevent the RFA-related complications and to increase the therapeutic efficacy of RFA, the choice of optimal operative approach should be based on the lesion size, number, and location and on the patient's clinical status. Hepatic inflow occlusion by Pringle maneuver during laparoscopic or open RFA therapy can reduce the blood loss and increase the therapeutic efficacy significantly.