ß-Thalassemia gene editing therapy: Advancements and difficulties.

ß-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of ß-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent ß-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of ß-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of ß-thalassemia diseases.

vEpiNet: A multimodal interictal epileptiform discharge detection method based on video and electroencephalogram data.

To enhance deep learning-based automated interictal epileptiform discharge (IED) detection, this study proposes a multimodal method, vEpiNet, that leverages video and electroencephalogram (EEG) data. Datasets comprise 24 931 IED (from 484 patients) and 166 094 non-IED 4-second video-EEG segments. The video data is processed by the proposed patient detection method, with frame difference and Simple Keypoints (SKPS) capturing patients' movements. EEG data is processed with EfficientNetV2. The video and EEG features are fused via a multilayer perceptron. We developed a comparative model, termed nEpiNet, to test the effectiveness of the video feature in vEpiNet. The 10-fold cross-validation was used for testing. The 10-fold cross-validation showed high areas under the receiver operating characteristic curve (AUROC) in both models, with a slightly superior AUROC (0.9902) in vEpiNet compared to nEpiNet (0.9878). Moreover, to test the model performance in real-world scenarios, we set a prospective test dataset, containing 215 h of raw video-EEG data from 50 patients. The result shows that the vEpiNet achieves an area under the precision-recall curve (AUPRC) of 0.8623, surpassing nEpiNet's 0.8316. Incorporating video data raises precision from 70% (95% CI, 69.8%-70.2%) to 76.6% (95% CI, 74.9%-78.2%) at 80% sensitivity and reduces false positives by nearly a third, with vEpiNet processing one-hour video-EEG data in 5.7 min on average. Our findings indicate that video data can significantly improve the performance and precision of IED detection, especially in prospective real clinic testing. It suggests that vEpiNet is a clinically viable and effective tool for IED analysis in real-world applications.

Role and Mechanism of the Renin-Angiotensin-Aldosterone System in the Onset and Development of Cardiorenal Syndrome.

Cardiorenal syndrome (CRS), a clinical syndrome involving multiple pathological mechanisms, exhibits high morbidity and mortality. According to the primary activity of the disease, CRS can be divided into cardiorenal syndrome (type I and type II), renal heart syndrome (type III and type IV), and secondary heart and kidney disease (type V). The renin-angiotensin-aldosterone system (RAAS) is an important humoral regulatory system of the body that exists widely in various tissues and organs. As a compensatory mechanism, the RAAS is typically activated to participate in the regulation of target organ function. RAAS activation plays a key role in the pathogenesis of CRS. The RAAS induces the onset and development of CRS by mediating oxidative stress, uremic toxin overload, and asymmetric dimethylarginine production. Research on the mechanism of RAAS-induced CRS can provide multiple intervention methods that are of great significance for reducing end-stage organ damage and further improving the quality of life of patients with CRS.

Mechanisms of Kidney Cell Pyroptosis in Chronic Kidney Disease and the Effects of Traditional Chinese Medicine.

Chronic kidney disease (CKD) is a major public health issue that is highly prevalent worldwide. Pyroptosis is an important pathological mechanism underlying kidney cell damage in CKD and is associated with the classic caspase-1-mediated pathway and nonclassic caspase-4/5/11-mediated pathway. The NLRP3-caspase-1-GSDMD signaling pathway is the key mechanism of kidney cell pyroptosis in CKD, and noncoding RNAs such as lncRNAs and miRNAs are important regulators of kidney cell pyroptosis in CKD. In addition, the NLRP1/AIM2-caspase-1-GSDMD and caspase-3-GSDME signaling pathways have also been shown to mediate kidney cell pyroptosis. Traditional Chinese medicine (TCM) and extracts can interfere with the occurrence and development of kidney cell pyroptosis in CKD by inhibiting the NLRP3 signaling pathway and oxidative stress, activating Nrf-2 signaling, protecting mitochondrial integrity, regulating AMPK signaling, and regulating TXNIP/NLRP3 axis, which have become increasingly prominent. It is critical to explore the effects of TCM on kidney cell pyroptosis in CKD and its mechanisms to identify targets and develop new and effective drugs.

Long noncoding RNA PVT1 promotes tumour progression via the miR-128/ZEB1 axis and predicts poor prognosis in esophageal cancer.

PURPOSE: To confirm the value of PVT1 as a prognostic marker both in tumour tissue and serum of patients with esophageal cancer and clarify the mechanism. METHODS: This study analyzed data obtained from 76 patients who were surgically treated from January 1, 2015, to December 31, 2016, and received a pathological diagnosis of ESCC. PVT1 levels in tumour tissue and serum were detected by qRT-PCR. Patient data were extracted from medical records, and follow-up evaluations were performed. The roles of PVT1 in proliferation, migration and invasion were by CCK-8 and Transwell in stable knockdown PVT1 cell lines. Signal pathways PVT1 promotes esophageal cancer were detected by qRT-PCR and western blot. RESULTS: PVT1 was overexpression in esophageal cancer tissues and high levels of PVT1 were correlated with lymphatic metastasis, high TNM stage and postoperative metastasis. High levels of PVT1 in tissues were correlated with worse metastasis-free survival (MFS) (HR: 2.578, 95% CI: 1.369-4.853). High level of PVT1 in serum was correlated with postoperative metastasis. High levels of PVT1 in serum were correlated with worse overall survival (OS) (HR: 2.124, 95% CI: 1.078-4.186) and worse MFS (HR: 2.786, 95% CI: 1.557-4.985). Knockdown of PVT1 decreased the cell proliferation, migration and invasion abilities of esophageal cancer cell lines. The expression of ZEB1 was significantly downregulated, and the expression of E-cadherin was increased by the knockdown of PVT1. Knockdown of miR-128 restored the altered proliferation, migration and invasion and the expression of ZEB1 and E-cadherin caused by knockdown of PVT1. CONCLUSIONS: High levels of PVT1 in serum were correlated with postoperative metastasis and a poor prognosis. PVT1 promoted ESCC progression via the miR-128/ZEB1/E-cadherin axis.

Effects of resveratrol in the signaling of neuropathic pain involving P2X3 in the dorsal root ganglion of rats.

Neuropathic pain is a major public health problem because it has a considerable impact on life quality of patients. Neuropathic pain caused by a lesion or disease of the somatosensory nervous system, which causes unpleasant and abnormal sensation (dysesthesia), an increased response to painful stimuli (hyperalgesia), and pain in response to a stimulus that does not normally provoke pain (allodynia). P2X receptors from dorsal root ganglion (DRG) play a crucial role in facilitating pain transmission at peripheral and spinal sites. Resveratrol (Res) has neuroprotective effects and improves the pathological and behavioral outcomes of various types of nerve injury. The present study examined the effects of Res on neuropathic pain. Neuropathic pain animal model was created by partial sciatic nerve ligation (pSNL) surgery. We found that consecutive intraperitoneal administration of Res for 21 days reduced the mechanical and thermal nociceptive responses induced by pSNL in a dose-dependent manner. Moreover, Res administration reversed P2X3 expression and phosphorylation of ERK in DRG neurons after peripheral nerve injury. Our results suggested that Res may ameliorate neuropathic pain by suppressing P2X3 up-regulation and ERK phosphorylation in DRG of neuropathic pain rats. Therefore, we concluded that Res has a significant analgesic effect on alleviating neuropathic pain, and thus may serve as a therapeutic approach for neuropathic pain.

A Consensus Framework of Distributed Multiple-Tilt Reconstruction in Electron Tomography.

The "missing wedge" of a single tilt in electron tomography introduces severe artifacts into the reconstructed results. To reduce the "missing wedge" effect, a widely used method is "multiple-tilt reconstruction," which collects projections using multiple axes. However, as the number of tilt series increases, the computing and memory costs also rise. The degree of parallelism is limited by the sample thickness, and a large memory requirement cannot be met by most multicore computers. In our study, we present a new fully distributed multiple-tilt simultaneous iterative reconstruction technique (DM-SIRT). To improve the parallelism of the reconstruction process and reduce the memory requirements of each process, we formulate the multiple-tilt reconstruction as a consensus optimization problem and design a DM-SIRT algorithm. Experiments show that in addition to slightly better resolution, DM-SIRT can obtain a 13.9 × accelerated ratio compared with the full multiple-tilt reconstruction version. It also has a 97% decrease in memory overhead and is 16 times more scalable than the full reconstruction version.

Molecular method for rapid detection of the red tide dinoflagellate Karenia mikimotoi in the coastal region of Xiangshan Bay, China.

The species Karenia mikimotoi is a common nearshore red tide alga that can secrete hemolytic exotoxin and ichthyotoxin, which can induce the death of fish and shellfish, causing severe economic losses. In this study, loop-mediated isothermal amplification (LAMP) was employed in combination with the lateral flow dipstick (LFD) visual detection method to establish the LAMP-LFD rapid detection method for K. mikimotoi. The internal transcribed spacer ITS1-5.8S-ITS2 of K. mikimotoi was used as the target sequence and was amplified with specific primers designed in this study. The results indicated that the amplification optimal reaction conditions for LAMP in this paper were for 20 min at 65 °C. Moreover, LAMP had excellent specificity, showing negative results for other common red tide causing algal species. In field samples, we successfully reduced the total time, with only 23 min needed from LAMP amplification to LFD result display, which was shorter than that of conventional PCR. Consequently, LAMP-LFD should be useful for rapid field detection of low-density K. mikimotoi and for the early prevention of red tide induced by such algae.

A Novel Chemoenzymatic Approach to Produce Cilengitide Using the Thioesterase Domain from Microcystis aeruginosa Microcystin Synthetase C.

Modern organic chemistry faces many difficulties in the reliable production of cyclopeptides, such as poor yields and insufficient regio- and stereoselectivity. Thioesterase (TE) shows impressive stereospecificity, region- and chemoselectivity during the cyclization of peptide substrates. The biocatalytic properties of TE provide high value for industrial applications. Herein, a novel chemoenzymatic method to synthesize cilengitide is described based on the cyclic activity of the TE domain from microcystin synthetase C (McyC) of Microcystis aeruginosa. In addition, a single active site mutation in the McyC TE was engineered to generate a more effective macrocyclization catalyst. Compared to the chemical approach to synthesize cilengitide, this novel enzyme-catalysed methodology exhibits a higher synthetic efficiency with an approximately 3.4-fold higher yield (49.2%).

A real-time recombinase polymerase amplification assay for the rapid detection of Vibrio harveyi.

Vibrio harveyi is a pathogen that infects fish and shellfish worldwide, causing severe economic losses for the aquaculture industry. As the early diagnosis of V. harveyi infection is crucial to disease surveillance and prevention in cultured marine animals, a fast and accurate method to detect V. harveyi is required. Here, we performed recombinase polymerase amplification (RPA) using novel primers specifically designed to recognize the V. harveyi toxR gene, which encodes a transmembrane protein, and then hybridized this gene with a carboxy fluorescein (FAM)-labeled probe. The optimal conditions for the real-time RPA assay were a probe concentration of 90 nM and a 20 min incubation at 37 °C. The sensitivity of our real-time RPA assay was 50 copies of the standard plasmid, while that of real-time PCR was 500 copies. In V. harveyi-spiked Pseudosciaena crocea samples, the sensitivity of our real-time RPA was 60 CFUs per reaction, while that of PCR was 600 CFUs per reaction. SPSS probit regression analysis indicated that the limit of detection (LOD) of our RPA assay, with 95% probability, was 18 copies. The LOD was reached within 20 min and was highly reproducible across eight independent assays. Our novel RPA method successfully differentiated V. harveyi from all other tested Vibrio species, including some that were closely related. Our real-time RPA assay, in combination with a rapid DNA extraction protocol, is a fast and accurate tool for the detection of V. harveyi and for monitoring disease outbreaks. This tool will be valuable for the aquaculture industry.

Recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of Salmonella in shellfish.

Salmonella is a major pathogen that causes acute foodborne outbreaks worldwide. Seafood, particularly shellfish, is a proven source of Salmonella spp. infection because many people prefer to eat it raw or lightly cooked. However, traditional identification methods are too time-consuming and complex to detect contamination of bacteria in the food chain in a timely manner, and few studies have aimed to identify Salmonella in shellfish early in the supply chain. We herein developed a method for rapid detection of Salmonella in shellfish based on the method of recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD), which targets the invasion gene A (invA). The RPA-LFD was able to function at 30-45 °C, and at the temperature of 40 °C, it only took 8 min of amplification to reach the test threshold of amplicons. The established method had both a good specificity and a sensitivity of 100 fg DNA per reaction (20 µL). Regarding practical performance, RPA-LFD performed better than real-time PCR. Another advantage of RPA-LFD is that it was capable of being performed without expensive equipments. Thus, RPA-LFD has potential for further development as a detection kit for Salmonella in shellfish and other foods under field conditions.

Amino acid-mediated aldolase immobilisation for enhanced catalysis and thermostability.

To improve the properties of the immobilised 2-deoxy-D-ribose-5-phosphate aldolase (DERA), unreacted functional groups on support surface were blocked with amino acids. The relative activities of the immobilised enzyme were 144.7 and 141.9% when the post-immobilisation modification was done with Arg and Phe, respectively. The residual activity of immobilised DERA after heating at 60 °C for 120 min was 65.1% when Phe and Val were used as the blocking amino acids, a 2.0- and 2.87-fold increase over that of the immobilised (no post-immobilisation blocking) and free DERA. Immobilised DERA maintained maximal activity in 2-deoxyribose-5-phosphate (DR5P) synthesis up to 600 mM of acetaldehyde, which was much higher than the amount of acetaldehyde tolerated by free enzyme (300 mM). This superior resistance to high acetaldehyde concentrations would accelerate the DR5P reaction by shifting the reaction equilibrium towards the product. The results from this study suggest that the novel immobilised DERA may be useful for industrial applications.

A novel and efficient method for the immobilization of thermolysin using sodium chloride salting-in and consecutive microwave irradiation.

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency.