In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

MSC-EXO and tempol ameliorate bronchopulmonary dysplasia in newborn rats by activating HIF-1α.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a major complication of premature infants and an important cause of morbidity and mortality. This study investigates the effect of the combination of mesenchymal stem cells-derived exosomes (MSC-EXO) and tempol on BPD and analyzes its mechanism. METHODS: MSC-EXO was extracted by centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis, and western blot analysis (WB). Tidal volume (TV), minute ventilation (MV), peak inspiratory flow (PIF), and dynamic pulmonary compliance (Cdyn) of rats were measured by BuxCo pulmonary function experimental platform. Hematoxylin-eosin staining was performed to observe the lung morphology and radical alveolar count (RAC) and mean linear intercept (MLI) were assessed. Immunofluorescence (IF) was conducted to detect the expression of CD31 and α-SMA in pulmonary blood vessels. The kits were used to calculate malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAOC) concentration in lung tissue. Enzyme linked immunosorbent assay was applied to detect the levels of IL-1ß, IL-17, IL-6, and IFN-γ in bronchoalveolar lavage fluid. In addition, the expressions of HIF-1α, vascular endothelial growth factor (VEGF), p-PI3K, and p-AKT were analyzed by WB and IF. RESULTS: We successfully extracted and identified MSC-EXO. In BPD rats, TV, MV, PIF, and Cdyn decreased, alveoli were simplified, and the number of interalveoli small vessels, blood vessel density decreased. Moreover, RAC, CD31, TAOC, and SOD decreased, and MLI, α-SMA, MDA, IL-1ß, IL-17, IL-6, and IFN-γ increased, which was reversed by the combination of MSC-EXO and tempol treatment after combined treatment. In addition, the expression levels of HIF-1α, VEGF, p-PI3K, and p-AKT were increased after combined treatment. CONCLUSIONS: Combined treatment could improve lung tissue injury, promote pulmonary vascular remodeling, restore lung function, and inhibit oxidative stress in BPD rats. These effects were achieved through activation of HIF-1α.

Effect of intermittent hypoxia or hyperoxia on lung development in preterm rat neonates during constant oxygen therapy.

Impaired lung development is a major negative factor in the survival of preterm neonates. The present study was aimed to investigate the impact of constant oxygen, intermittent hyperoxia, and hypoxia on the lung development in preterm rat neonates. Neonatal rats were exposed to 40% O2 with or without brief hyperoxia episodes (95% O2 ) or brief hypoxia episodes (10% O2 ) from day 0 to day 14, or to room air. The body weight, radical alveolar count (RAC), and total antioxidant capacity (TAOC) were significantly lower whereas the lung coefficient and malondialdehyde (MDA) were significantly higher in the hyperoxia and hypoxia groups than the air control and constant oxygen group at day 7, day 14, and day 21 after birth. The lung function indexes were reduced by intermittent hyperoxia and hypoxia. In contrast, the constant oxygen therapy increased the lung function. HIF-1α and VEGF expression were significantly increased by hypoxia and decreased by hyperoxia. The constant oxygen therapy only decreased the HIF-1α expression at day 14 and 21. In summary, the constant oxygen treatment promoted lung function without affecting the antioxidative capacity in preterm rat neonates. While intermittent hyperoxia and hypoxia inhibited lung development, decreased antioxidative capacity, and dysregulated HIF-1α/VEGF signaling in preterm rat neonates.

Congenital brucellosis in a Chinese preterm neonate: A case report.

Although extremely rare, congenital brucellosis can occur via perinatal transmission. We report a case of an infant born prematurely at 34-36 weeks' gestation who had pyrexia, shortness of breath, hepatosplenomegaly and thrombocytopenia. Blood cultures were positive for Gram-negative coccobacilli and Brucella infection was suspected. While, serological tests were negative for Brucella antibodies, B. melitensis infection was confirmed by polymerase chain reaction (PCR). Serology of the parents' blood confirmed the presence of Brucella. The family did not live in an endemic area but had ridden a camel 12 months before the pregnancy. The bacteria may have been sexually transmitted from father to mother and then to foetus via an intrauterine infection. In endemic areas or where the family has been in close contact with infected animals, brucellosis should be suspected in a severely ill neonate with an unknown infection. Thorough medical histories from the family are essential as early diagnosis and prompt therapy will almost certainly improve neonatal outcome.

Long range haplotyping of paired-homologous chromosomes by single-chromosome sequencing of a single cell.

The longest possible haplotype is chromosome haplotype that is a set of co-inherited alleles occurred on a single strand chromosome inherited from one parent. Standard whole-genome shotgun sequencing technologies are limited by the inability to independently study the haplotype of homologous chromosomes due to the short-reads sequencing strategy and disturbance of homologue chromosomes. Here, we investigated several types of chromosomal abnormalities by a dilution-based method to separate an intact copy of homologous chromosome from human metaphase cells, and then single chromosomes were independently amplified by whole-genome amplification methods, converted into barcoded sequencing libraries, and sequenced in multiplexed pools by Illumina sequencers. We analyzed single chromosome derived from single metaphase cells of one patient with balanced chromosomal translocation t(3;5)(q24;q13), one patient with (47, XXY) karyotype and one with (47, XY, 21+) Down syndrome. We determined the translocation region of chromosomes in patient with t(3;5)(q24;q13) balanced chromosomal translocation by shallow whole-genome sequencing, which is helpful to pinpoint the chromosomal break point. We showed that SCS can physically separate and independently sequence three copies of chromosome 21 of Down syndrome patient. SCS has potential applications in personal genomics, single-cell genomics, and clinical diagnosis, particularly in revealing chromosomal level of genetic diseases.

[Influence of intrauterine hypoxia on the lung blood vessel development in rats after birth and expression of VEGF in the lung].

OBJECTIVE: To observe the effect of intrauterine hypoxia on the development of rat lung after birth under ordinary pressure and normoxia, on the expression of vascular endothelial growth factor (VEGF) in the lung as the age increasing after birth, and to provide experimental basis for the treatment of intrauterine hypoxia after baby was born. METHODS: Intrauterine hypoxia models were established. The rats were divided into an air-control group (the control group) and a hypoxic 6-day group (the hypoxic group). All rats were fed under normal pressure and normoxia after they were born. At postnatal 7, 14, and 21 days, we measured the pulmonary vascular morphometry, detected the expression of VEGF protein with immunohistochemisty, the expression of VEGF mRNA with real-time PCR, and observed the alteration of capillary endothelium in the lung tissues under the electron microscope. RESULTS: The expression of VEGF protein and VEGF mRNA in the 2 groups increased as the rats grew, but the expression increased slower in the hypoxic group than that in the control group. The increase curve of the 2 groups crossed. There was no significant difference between the 2 groups in the pulmonary vascular morphometry at each experiment time point. Hyperplasia of capillary endothelium decreased with age. Cellular edema of capillary endothelium was obvious especially at the 14th day after birth under the electron microscope. CONCLUSION: The expression of VEGF protein and VEGF mRNA has slower increase in the intrauterine hypoxic rats than that in the normal control rats. The expression of VEGF may influence the development of lung vessel after rats was born.