3D-printed microfluidic device for high-throughput production of lipid nanoparticles incorporating SARS-CoV-2 spike protein mRNA.

Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL min-1 range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL min-1) of LNPs compared to commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (>90%). The 3D-printed device provides a cost-effective and easily prepared solution for high-throughput LNP production.

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1.

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.

Compatibility of Popular Three-Dimensional Printed Microfluidics Materials with In Vitro Enzymatic Reactions.

3D printed microfluidics offer several advantages over conventional planar microfabrication techniques including fabrication of 3D microstructures, rapid prototyping, and inertness. While 3D printed materials have been studied for their biocompatibility in cell and tissue culture applications, their compatibility for in vitro biochemistry and molecular biology has not been systematically investigated. Here, we evaluate the compatibility of several common enzymatic reactions in the context of 3D-printed microfluidics: (1) polymerase chain reaction (PCR), (2) T7 in vitro transcription, (3) mammalian in vitro translation, and (4) reverse transcription. Surprisingly, all the materials tested significantly inhibit one or more of these in vitro enzymatic reactions. Inclusion of BSA mitigates only some of these inhibitory effects. Overall, inhibition appears to be due to a combination of the surface properties of the resins as well as soluble components (leachate) originating in the matrix.

[Diversity and community characteristics of organic phosphate-mineralizing bacteria in the sea area between Minjiang Estuary to Pingtan.] / 闽江口-å¹³æ½­æµ·åŸŸæœ‰æœºè§£ç£·èŒå¤šæ ·æ€§åŠç¾¤è½ç‰¹å¾.

Microbial mineralization of organic phosphorus is an important component of marine phosphorus cycle. The research on organic phosphate-mineralizing bacteria (OPB) is helpful to reveal microbial driving mechanism of organic phosphorus mineralization in eutrophic sea area. The diversity and community characteristics of OPB were examined by Illumina high-throughput sequencing using the primer sets phoX in the sea area between Minjiang Estuary to Pingtan in April (spring) and July (summer) 2019. The results showed that the Shannon index of OPB in the surface seawater samples ranged from 3.21 to 7.91, and that the diversity at each station was greater in spring than that in summer. Shannon index of OPB in the sediment samples ranged from 2.04 to 8.70, which was greater in summer than that in spring. Shannon index of OPB in surface seawater of each station was higher than that of sediment in spring, while it was in adverse in summer. Nine phyla of OPB were detected in surface seawater, with Proteobacteria and Cyanobacteria being the most abundant. Tweleve phyla of OPB were detected in the sediments, with Proteobacteria and Bacteroidetes being the most dominant. OPB community composition at the genus level showed obvious spatio-temporal variation. Leisingera, Phaeobacter, Thalassococcus, and Pseudomonas were the major genera in the seawater in spring, while Synechococcus, Halioglobus, Roseovarius, Phaeo-bacter, Sulfitobacter, and Hyphomonas were the major genera detected in summer. Leisingera, Phaeobacter, Vibrio, and Sulfitobacter were major genera in the sediment in spring, while Azospirillum, Aminobacter, Sulfurifustis, Burkholderia, and Thiohalobacter were the major genera in summer. A large number of unclassified OPB were detected in both surface seawater and sediment. The redundancy analysis results showed that dissolved oxygen, water temperature, pH, dissolved inorganic nitrogen, NO2--N, and NO3--N had great influences on community distribution of OPB in the surface seawater. The abundant OPB in the surface seawater and sediment might play an important role in phosphorus cycle in this sea area.

Enabling Flow-Based Kinetic Off-Rate Selections Using a Microfluidic Enrichment Device.

Modern genomic sequencing efforts are identifying potential diagnostic and therapeutic targets more rapidly than existing methods can generate the peptide- and protein-based ligands required to study them. To address this problem, we have developed a microfluidic enrichment device (MFED) enabling kinetic off-rate selection without the use of exogenous competitor. We tuned the conditions of the device (bed volume, flow rate, immobilized target) such that modest, readily achievable changes in flow rates favor formation or dissociation of target-ligand complexes based on affinity. Simple kinetic equations can be used to describe the behavior of ligand binding in the MFED and the kinetic rate constants observed agree with independent measurements. We demonstrate the utility of the MFED by showing a 4-fold improvement in enrichment compared to standard selection. The MFED described here provides a route to simultaneously bias pools toward high-affinity ligands while reducing the demand for target-protein to less than a nanomole per selection.

[Community characteristics of polyphosphate accummulating organisms in the heart sediment of three reservoirs in Fujian Province, China.] / 福建省3座水库库心沉积物聚磷菌的群落特征.

Polyphosphate accummulating organisms (PAOs) play an important role in the phosphorus metabolic cycling in the sediment of reservoir. We assessed the diversity and community structure of PAOs in the sediments by T-RFLP and clone sequencing which targeted ppk1 gene at the hearts of three reservoirs (Jiulongjiangxipi reservoir, Sanshiliujiao lake reservoir and Dongyaxi reservoir) in Fujian Province. The results showed that the diversity of PAOs varied among different reservoirs, though not statistically significant. The diversity of PAOs in the Sanshiliujiao lake reservoir was highest (Shannon index H=2.89±0.03, Simpson index D=0.06±0.01). The community structure of the PAOs in the Sanshiliujiao lake reservoir was most complicated, consistent with the results of the T-RFLP. The differences of dominant PAOs genera in three reservoirs were distinct, mainly concentrated in the Proteobacteria, Actinobacteria and Acidobacteria. The percentage of those three phylum accounted for 74.5%, 85.0% and 75.0%, respectively, of the total PAOs. The dominant groups in each reservoir sediment were Anaeromyxobacter and Solibacter. Various forms of phosphorus had certain influence on the diversity of PAOs. There were significantly correlation between Fe/Al-P and PAOs diversity and community structure. The dominant genus in the three reservoirs, Anaeromyxobacter, was positively correlated with all forms of phosphorus and significantly correlated with insoluble phosphorus such as OP and Ca-P, while Solibacter was negatively correlated with all forms of phosphorus. The results suggested that PAOs had important impacts on the phosphorus cycle of sediment in eutrophicatied reservoirs.

Liposome production and concurrent loading of drug simulants by microfluidic hydrodynamic focusing.

Liposomes are spherical vesicles enclosed by phospholipid bilayers. Nanoscale liposomes are widely employed for drug delivery in the pharmaceutical industry. In this study, nanoscale liposomes are fabricated using the microfluidic hydrodynamic focusing (MHF) approach, and the effects of flow rate ratio (FRR) on liposome size and drug loading efficiency are studied. Fluorescein isothiocyanate modified dextran is used as a hydrophilic drug simulant and Nile red is used as a hydrophobic drug simulant. The experiment results show that hydrophilic drug simulant loading efficiency increases as FRR increases and eventually plateaues at around 90% loading efficiency. The hydrophobic drug simulant loading efficiency and FRR have a positive linear correlation when FRR varies from 10 to 50. Concurrent loading of both hydrophilic and hydrophobic drug simulants maintains the same loading efficiencies as those of loading each drug simulant alone. A negative correlation between liposome size and FRR is also confirmed. Unloaded liposomes and hydrophilic drug-loaded liposomes are of the same sizes, and are smaller than the ones loaded with the hydrophobic drug simulants alone or combined. The results suggest tunable liposome size and drug loading efficiency with the MHF technique. This provides evidence to encourage further studies of microfluidic liposome fabrication in the pharmaceutical industry.

The mPEG-PCL Copolymer for Selective Fermentation of Staphylococcus lugdunensis Against Candida parapsilosis in the Human Microbiome.

Many human skin diseases, such as seborrheic dermatitis, potentially occur due to the over-growth of fungi. It remains a challenge to develop fungicides with a lower risk of generating resistant fungi and non-specifically killing commensal microbes. Our probiotic approaches using a selective fermentation initiator of skin commensal bacteria, fermentation metabolites or their derivatives provide novel therapeutics to rein in the over-growth of fungi. Staphylococcus lugdunensis (S. lugdunensis) bacteria and Candida parapsilosis (C. parapsilosis) fungi coexist in the scalp microbiome. S. lugdunensis interfered with the growth of C. parapsilosis via fermentation. A methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) (mPEG-PCL) copolymer functioned as a selective fermentation initiator of S. lugdunensis, selectively triggering the S. lugdunensis fermentation to produce acetic and isovaleric acids. The acetic acid and its pro-drug diethyleneglycol diacetate (Ac-DEG-Ac) effectively suppressed the growth of C. parapsilosis in vitro and impeded the fungal expansion in the human dandruff. We demonstrate for the first time that S. lugdunensis is a skin probiotic bacterium that can exploit mPEG-PCL to yield fungicidal short-chain fatty acids (SCFAs). The concept of bacterial fermentation as a part of skin immunity to re-balance the dysbiotic microbiome warrants a novel avenue for studying the probiotic function of the skin microbiome in promoting health.