Sheathless microflow cytometer utilizing two bulk standing acoustic waves.

In recent years, microflow cytometry has become a popular research field because of its potential to provide low-cost and disposable chips for complex cell analyses. Herein, we demonstrate a sheathless microflow cytometer which integrates a bulk standing acoustic wave based microchip capable of three dimensional cell focusing. Flow cytometry was successfully demonstrated using this system with a coefficient of variation (CV) of 2.16% with standard calibration beads. The sensitivities calibrated by rainbow beads are 518 MEFL in fluorescein Isothiocyanate (FITC) channel and 264 MEPE in P-phycoerythrin (PE) channels, respectively. The linearities are more than 99% in both channels. The capability of the proposed microflow cytometer is further demonstrated by immunologically labeled leukocytes differentiation in blood. This acoustic-based microflow cytometer did not require any sheath flows or complex structures and can be mass produced. Because of the simple fluid channel, the chip can be easily made pipeless, disposable for applications requiring no cross contamination. Moreover, with the gentle and bio-compatible acoustic waves used, this technique is expected to maintain the viability of cells and other bioparticles.

Osteoblast-derived extracellular matrix coated PLLA/silk fibroin composite nanofibers promote osteogenic differentiation of bone mesenchymal stem cells.

Poly-L-lactic acid (PLLA) is one of the most commonly used synthetic materials for regenerative medicine, and silk fibroin (SF) is a natural protein with excellent biocompatibility. Combination of PLLA and SF in a proper proportion by electrospinning may generate composite nanofibers that could meet the requirements of scaffolding in bone tissue engineering. The application of PLLA/SF nanofibrous scaffold for osteogenesis is well established in vitro and in vivo. However, PLLA/SF nanofibrous scaffold does not have an ideal ability to promote cell adhesion, proliferation, and differentiation. Extracellular matrix (ECM) plays a critical role in modulating cellular behavior. However, the role of combination of natural ECM with nanofibrous scaffold in regulating osteogenic differentiation is unclear. In this study, we aimed to develop a novel composite PLLA/SF nanofibrous scaffold coated with osteoblast-derived extracellular matrix (O-ECM/PLLA/SF) and analyze the effects of the modified scaffold on osteogenic differentiation of BMSCs. The surface structural features and compositions of the O-ECM/PLLA/SF scaffold were characterized by SEM and immunofluorescence staining. The capacities of the O-ECM/PLLA/SF scaffold to induce osteogenic differentiation of BMSCs were investigated by alkaline phosphatase (ALP) and alizarin red staining (ARS). The results showed BMSCs cultured on O-ECM/PLLA/SF scaffold significantly increased osteogenic differentiation compared with cells cultured individually on a scaffold or O-ECM. Collectively, these findings indicate that O-ECM-coated nanofibrous scaffold can be a promising strategy for osteogenic differentiation of BMSCs, opening a new possibility of utilizing composite scaffolds for bone tissue engineering.

A university-clustered tuberculosis outbreak during the COVID-19 pandemic in eastern China.

During the COVID-19 pandemic in 2020, a tuberculosis outbreak occurred in a university in eastern China, with 4,488 students and 421 staff on the campus. A 19-year-old student was diagnosed in August 2019. Later, the first round of screening was initiated among close contacts, but no active cases were found. Till September 2020, four rounds of screening were performed. Four rounds of screening were conducted on September 9, November 8, November 22-25 in 2019 and September 2020, with 0, 5, 0 and 43 cases identified, respectively. A total of 66 active tuberculosis were found in the same university, including 4 sputum culture-positive and 7 sputum smear-positive. The total attack rate of active tuberculosis was 1.34% (66/4909). The whole-genome sequencing showed that the isolates belonged to the same L2 sub-specie and were sensitive to all tested antituberculosis drugs. Delay detection, diagnosis and report of cases were the major cause of this university tuberculosis epidemic. More attention should be paid to the asymptomatic students in the index class. After the occurrence of tuberculosis cases in schools, multiple rounds of screening should be carried out, and preventive therapy should be applied in a timely manner.

Optimal design of a driver of interdigital transducers used to generate standing surface acoustic waves for cell sorting.

A compact driver based on current feedback amplifiers is designed to drive interdigital transducers (IDTs) that generate standing surface acoustic waves for cell sorting. Compared with commercial RF amplifiers, this driver can be used to drive a wider range of loads without impedance matching. Furthermore, the driver works in a switch mode triggered by target cells, which significantly reduces power consumption in the system. A Butterworth-Van Dyke equivalent circuit was fabricated to study the electrical characteristics of the IDTs, and the driver was designed and optimized by circuit simulations. A cell sorter was constructed and tested experimentally to demonstrate that the driver meets sorting requirements. The driver allows the cell sorter to extract rare cells while otherwise consuming low power.

Influence of 24-diamino-5-phenylthiazole on neomycin ototoxicity in cultured organ of Corti explants.

Hair cells do not undergo spontaneous regeneration when they are damaged in the mammalian organ of Corti, leading to irreversible hearing loss. Previous studies have shown that 24-diamino-5-phenylthiazole (DAPT), an inhibitor of Notch signaling, plays a major role in inner ear development. However, whether DAPT influences antibiotic-induced hair cell damage remains uncertain. The present study aimed to investigate whether DAPT exerts protective or regenerative effects on neomycin-damaged hair cells. A histological analysis was carried out to assess the number and morphological changes of hair cells in cultured organ of Corti explants. Our results showed that in-vitro treatment with DAPT induced extra hair cells, whereas no newly generated supporting cells were found. We also found that DAPT was effective for preventing hair cell loss when cotreatment with neomycin was performed, suggesting that DAPT exerted protective effects on neomycin ototoxicity. In addition, DAPT treatment for 2-4 days following neomycin damage induced supernumerary hair cells. These findings indicate that inhibition of Notch signaling is a possible strategy for the treatment of hair cell loss caused by aminoglycoside antibiotics.

[Cloning and polymorphism analysis of intron 2 of the GH gene in Takifugu].

Size and nucleotide polymorphisms of intron 2 of the growth hormone (GH) gene was studied in 82 individuals from Takifugu obscurus, Takifugu rubripes and Takifugu niphobles with PAGE, SSCP and cloning. Results showed that there were nine length variants (A~I) in these fishes, ranging from 271351 bp, with a frequency of variation of 24.22%. Sequence analysis of the 9 length variants showed the average percentages of the four bases (ATG and C) were 17.15, 20.77, 37.38 and 24.70, respectively, and the difference between GC content (45.47) and AT content (54.53) was not remarkable. Length variation was mainly due to the variable number of microsatellite TCTG repeats (N=20 approximately 40). Further sequence comparison revealed 4 substitution sites: a (C-->A) transversion at nucleotide 103 and 3 transitions 83(C-->T), 101(A-->G) and 296(G-->A), respectively. Phylogenetic analysis of these length variants placed DD and II into one group first, which was subsequently joined by GG and AA, BB and CC, EE and HH were then clustered together, FF was joined last. Thus, intraspecific variations were much smaller than interspecific variations for the intron 2 of the GH gene.

[Experimental study of protective effects of Xuebijing injection on stress-induced organ damage in rabbit].

OBJECTIVE: To investigate the protective effects of the integrated traditional Chinese and western medicine Xuebijing injection on stress-induced organ damage in rabbits. METHODS: Forty rabbits were randomly divided into four groups: the control group, the model group, the western medicine group, and the Xuebijing group. The stress-induced organ damage model was replicated by soak the rabbits in water, the animals in the western medicine group and Xuebijing group received injection of lytic cocktail and Xuebijing, respectively. The changes in cortisol (Cor), thromboxane A(2) (TXA(2)), endothelin (ET), nitric oxide synthase (NOS) were determined at different time points in all the groups. The pathologic changes of the gastric mucosa, the adrenal gland and the cardiac muscle cell were observed. RESULTS: The content of Cor increased significantly in model group (P<0.01). The content of Cor decreased in the western medicine group and Xuebijing group, the changes showed no significant difference between two groups (P>0.05). The contents of ET, TXA(2) decreased and NOS increased in Xuebijing group compared with the western medicine group, the differences were significant (all P<0.05). The pathological changes of the gastric mucosa, the adrenal gland and the cardiac myocyte were less marked in Xuebijing group, compared with the western medicine group, the difference was significant (P<0.05). CONCLUSION: Xuebijing has better protective effects on stress-induced organ damage.

[Preparation of chicken red blood cells for calibration of flow cytometry].

OBJECTIVE: To prepare stable chicken red blood cells for the calibration of flow cytometry. METHODS: The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells. RESULTS: Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01]. CONCLUSION: The chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.