A microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from low copy/single bacterial cells in minute sample volume followed by direct on-chip quantitative PCR assay.

Purification of nucleic acids from a low quantity of bacterial cells in minute volume is important in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cells in the range of 5000 down to a single cell in the sample volume of 1 µl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of microwells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the microwell array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved using the chip-based liquid phase nucleic acid purification technique compared to that obtained by the conventional column-based solid phase nucleic acid extraction method. One step vacuum-driven microfluidics allowed an on-chip quantitative PCR assay to be carried out in the same microwells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purification device set in a two-dimensional (2D) array format of 900 microwells, it was demonstrated for the first time that high-throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.

Molecular mechanism of ensitrelvir inhibiting SARS-CoV-2 main protease and its variants.

SARS-CoV-2 poses an unprecedented threat to the world as the causative agent of the COVID-19 pandemic. Among a handful of therapeutics developed for the prevention and treatment of SARS-CoV-2 infection, ensitrelvir is the first noncovalent and nonpeptide oral inhibitor targeting the main protease (Mpro) of SARS-CoV-2, which recently received emergency regulatory approval in Japan. Here we determined a 1.8-Å structure of Mpro in complex with ensitrelvir, which revealed that ensitrelvir targets the substrate-binding pocket of Mpro, specifically recognizing its S1, S2, and S1' subsites. Further, our comprehensive biochemical and structural data have demonstrated that even though ensitrelvir and nirmatrelvir (an FDA-approved drug) belong to different types of Mpro inhibitors, both of them remain to be effective against Mpros from all five SARS-CoV-2 variants of concern, suggesting Mpro is a bona fide broad-spectrum target. The molecular mechanisms uncovered in this study provide basis for future inhibitor design.

DPP-4 inhibitor saxagliptin ameliorates oxygen deprivation/reoxygenation-induced brain endothelial injury.

Cardiovascular diseases are the main cause of death and disability among diabetes patients. Atherosclerosis-associated stroke is one of the most severe complications in diabetes patients. DPP-4 inhibitors are a class of potent anti-glycemic agents used to treat diabetes. Recently, some DPP-4 inhibitors have been shown to have cardiovascular benefits. In this study, we reveal that saxagliptin, one of the most widely used DPP-4 inhibitors, exhibits vascular protective effects against oxygen and glucose depletion/reoxygenation (OGD/R) in human brain vascular endothelial cells. Our data show that DPP-4 is fairly expressed in brain endothelial cells and its expression is induced by OGD/R. The results of MTT assay show that inhibition of DPP-4 by saxagliptin ameliorates OGD/R-induced reduced cell viability, and LDH assay demonstrated that saxagliptin reduces cellular toxicity. Furthermore, we show that saxagliptin mitigates OGD/R-induced collapse of mitochondrial membrane potential (MMP). Saxagliptin also reduces oxidative stress-induced release of 4-HNE and the NAPDH oxidase catalytic subunit NOX-4. At the molecular level, saxagliptin suppresses OGD/R-induced expression of pro-inflammatory cytokines and production of vascular adhesion molecules including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, monocyte chemoattractant protein 1 (MCP-1), vascular cellular adhesion molecule 1 (VCAM-1), and E-selectin. Mechanistically, saxagliptin inhibits activation of the NF-κB pathway by OGD/R via its inhibitory effect on nuclear p65 and NF-κB promoter activity. Collectively, our study explicitly demonstrates the cellular protective effect of saxagliptin against OGD/R-induced brain endothelial injury. Our findings extend our recognition of the protective roles of DPP-4 inhibitors in brain vascular cells.

Anagliptin protects neuronal cells against endogenous amyloid ß (Aß)-induced cytotoxicity and apoptosis.

Excessive generation and accumulation of amyloid-ß (Aß) fragments by familial mutations of amyloid precursor protein (APP) and presenilin 1 (PS1) play a key role in causing oxidative stress, mitochondrial abnormalities and neuronal apoptosis in Alzheimer's disease (AD). Anagliptin, a novel DPP-4 inhibitor, is a clinical drug for the management of type II diabetes approved for use in 2012. Little on the pharmacological function of anagliptin against Aß-induced cytotoxicity in neuronal cells is known. Here, we examined the protective capacities of anagliptin against cytotoxicity in N2a neuronal cells overexpressing APP Swedish mutant and PS1 exon 9 deletion mutant (N2a/Swe.D9). Our results demonstrate that anagliptin reduced the production of ROS and the expression of NADPH oxidase 4 (NOX-4) in N2a/Swe.D9 cells. We also reported that anagliptin activates the antioxidant system by increasing the level of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity. Notably, anagliptin is able to improve mitochondrial function by elevating mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production. Additionally, our results demonstrate that anagliptin decreased the vulnerability of cells to hydrogen peroxide (H2O2)-induced secondary insult by increasing cell viability and reducing the secretion of lactate dehydrogenase (LDH) and high mobility group box 1 protein (HMGB-1). Importantly, we found that treatment with anagliptin suppressed the mitochondrial-dependent apoptosis pathway by preventing the translocation of cytochrome C, reducing cleavage of caspase-3, and the inhibiting expression of Bax. These results implicate that anagliptin may have potential as a therapeutic agent for AD treatment.

[Efficacy evaluation of specific immunotherapy with standardized house dust mite allergen preparation for allergic rhinitis].

OBJECTIVE: To evaluate the efficacy of specific immunotherapy (SIT) with standardized house dust mite allergen preparation for allergic rhinitis (AR). METHOD: Fifty-five patients with allergic rhinitis caused by house dust mites were selected in this self-control study. Clinical efficacy was evaluated by symptom and sign score after two years of specific immunotherapy and compared with pre-treatment scores. RESULT: After completing the study, a clinically significant reduction in symptom and sign score in these patients was noted, compared with that of pretreatment. And the difference was statistically significant (P < 0.01). CONCLUSION: Standardized house dust mite allergen preparation is an effective treatment in patients suffering from allergic rhinitis due to house dust mites.

[Value of nasal endoscopy in the surgery of the nasal inverted papillomas].

OBJECTIVE: To evaluate the curative effect of endoscopic surgery (ESS) in the treatment of inverted papillomas in the nasal cavity and paranasal sinus. METHOD: We report on 16 clinicopathologic cases of inverted papilloma. After preoperative endoscopic examination and CT screening, endoscopic sinus surgery was performed under general anesthesia. RESULT: The follow-up period ranged between one year to two years. Recurrence was observed in 2 cases. CONCLUSION: The nasal endoscopy is a better method for treating Krouse I-II stage nasal inverted papilloma. The advantages of endoscopic surgery include clear visual field, less bleeding, limited reoccurrence, and avoidance of facial incision.