Gapless biosynthetic pathway enables sustainable paclitaxel production.

A recent leading-edge study by Jiang et al. identified two enzymes that are responsible for key reactions in the biosynthesis of baccatin III. The authors successfully reconstructed the baccatin III synthesis pathway with a minimal number of synthetic enzymes in tobacco leaves, laying the foundation for industrial-scale sustainable production of the anticancer drug paclitaxel.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy.

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Δ Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles.

Correlated analysis of multiple biochemical parameters at the single-particle level and in a high-throughput manner is essential for insights into the diversity and functions of biological nanoparticles (BNPs), such as bacteria and subcellular organelles. To meet this challenge, we developed a highly sensitive spectral nano-flow cytometer (S-nFCM) by integrating a spectral recording module to a laboratory-built nFCM that is 4-6 orders of magnitude more sensitive in side scattering detection and 1-2 orders of magnitude more sensitive in fluorescence detection than conventional flow cytometers. An electron-multiplying charge-coupled device (EMCCD) was used to acquire the full fluorescence spectra of single BNPs upon holographic grating dispersion. Up to 10,000 spectra can be collected in 1 min with 2.1 nm resolution. The precision, linearity, and sensitivity were examined. Complete discernment of single influenza viruses against the background signal, discrimination of different strains of marine cyanobacteria in a mixed sample based on their spectral properties of natural fluorescence, classification of bacterial categories exhibiting different patterns of antigen expression, and multiparameter analysis of single mitochondria for drug discovery were successfully demonstrated.

Investigation of enzymatic quality and quantity using pyridoxal 5'-phosphate (PLP) regeneration system as a decoy in Escherichia coli.

Pyridoxal 5'-phosphate (PLP), an essential cofactor for multiple enzymes, was used as a protein decoy to prompt enzyme expression and activity for the first time. The best chassis, denoted as WJK, was developed using a pyridoxal kinase (PdxK) and integrated at the HK022 phage attack site of Escherichia coli W3110. When compared with the original strain, the amount and activity of lysine decarboxylase (CadA) in WJK were significantly increased by 100 % and 120 %, respectively. When supplementary nineteen amino acids as second carbon source, cell growth and protein trade-off were observed. The transcriptional levels of genes from glycolysis to TCA cycle, adhE, argH and gdhA were dominating and redirected more flux into α-ketoglutarate, thus facilitated cell growth. Stepwise improvement was conducted with pyridoxal and nitrogen-rich medium; hence, CadA activity was increased to 60 g-cadaverine/g-dry cell weight/h. By reutilizing the whole-cell biocatalysts in two repeated reactions with the supplementation of fresh cells, a total cadaverine of 576 g/L was obtained even without additional PLP. Notably, PLP decoy augment the enzymatic activities of 5-aminolevulinic acid synthase and glutamate/lysine/arginine decarboxylases by over 100 %. Finally, a conserved PLP-binding pocket, Ser-His-Lys, was identified as a vital PLP sponge site that simultaneously improved protein quality and quantity.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

High-Throughput Counting and Sizing of Therapeutic Protein Aggregates in the Nanometer Size Range by Nano-Flow Cytometry.

Protein aggregation is one of the greatest challenges in biopharmaceuticals as it could decrease therapeutic efficacy, induce immunogenicity, and reduce shelf life of protein drugs. However, there lacks high-throughput methods than can count and size protein aggregates in the nanometer size range, especially for those smaller than 100 nm. Employing a laboratory-built nano-flow cytometer (nFCM) that enables light scattering detection of single silica nanoparticles as small as 24 nm with sizing resolution and accuracy comparable to those of electron microscopy, here, we report a new benchmark to analyze single protein aggregates as small as 40 nm. With an analysis rate of up to 10,000 particles/min, the size distribution and particle concentration of nanometer protein aggregates can be acquired in 2-3 min. Employing heat-induced aggregation of bovine serum albumin (BSA) at high concentrations as the model system, effects of different categories of excipients, including sugars, polyols, salts, and amino acids on the inhibition of protein aggregation were investigated. Strikingly enough, as high as 1010 to 1012 particles/mL of protein aggregates were observed in the size range of 40 to 200 nm for therapeutic proteins of human serum albumin injection, reconstituted recombinant human interieukin-2 solution, and human immunoglobulin injection. nFCM opens a new avenue to count and size nanometer protein aggregates, suggesting its future usability in the quality assessment and formulation promotion of therapeutic proteins.

Refractive Index Determination of Individual Viruses and Small Extracellular Vesicles in Aqueous Media Using Nano-Flow Cytometry.

The refractive index (RI) is a fundamental physical property of materials. Although measurement of the RI of biological nanoparticles (BNPs) in aqueous media is of great importance to basic research and biomedical applications, it is hampered by their tiny size, large intrinsic heterogeneity, and weak scattering. Here, we report the development of a label-free technique that can determine the RI of individual viruses and small extracellular vesicles (sEVs) with high precision and an analysis rate up to 10 000 particles per minute. This was achieved via the combination of high-sensitivity light-scattering detection by nanoflow cytometry (nFCM) and the Mie theory calculation. With the measured RIs for T7 virions, T7 capsids, and sEVs, the concentrations of nucleic acid in viral particles and protein in the lumen of sEVs were estimated. Furthermore, building upon a simplified core-shell model, the RIs of sEVs ranging from 40 to 200 nm were obtained. By using these RIs, a statistically robust size distribution of sEVs was acquired in minutes with accuracy and resolution matched closely with those of cryo-TEM measurements. Our approach could become an important tool in the RI determination of single BNPs.

Noninvasive Diagnosis of Nasopharyngeal Carcinoma Based on Phenotypic Profiling of Viral and Tumor Markers on Plasma Extracellular Vesicles.

Nasopharyngeal carcinoma (NPC) is a malignant tumor commonly associated with Epstein-Barr virus (EBV) infection, and its early diagnosis as well as its differentiation from nasopharyngitis (NPG) remains challenging due to the insufficient sensitivity of routine screening methods in clinical practice. To date, circulating extracellular vesicles (EVs, 40-1000 nm) have shown appealing potential in liquid biopsy for cancer diagnosis and prognosis. Herein, nanoflow cytometry (nFCM) capable of single EV analysis was applied to examine the expression of surface proteins with very low copy numbers on individual EVs as small as 40 nm. The particle concentrations of five EV subsets exposing EBV-encoded latent membrane proteins (LMP1 and LMP2A) and tumor markers (PD-L1, EGFR, and EpCAM) in plasma were determined rapidly via single-particle enumeration. We identified a five-marker panel named EVSUM5 (an unweighted sum of the concentration of the five individual EV subsets) that significantly surpassed the traditional VCA-IgA assay in discriminating NPC patients from both healthy donors and NPG patients with accuracies of 96.3 and 83.1%, respectively. Moreover, EVSUM2 (an unweighted sum of virus-specific LMP1- and LMP2A-positive EVs) could achieve the diagnosis of NPG with an accuracy of 82.6%. Collectively, the work presented a rapid, reliable, and noninvasive method as well as two diagnostic markers to help more accurately differentiate NPC from NPG patients and healthy donors in clinical practice.

Analysis of extracellular vesicle DNA at the single-vesicle level by nano-flow cytometry.

It has been demonstrated recently that extracellular vesicles (EVs) carry DNA; however, many fundamental features of DNA in EVs (EV-DNA) remain elusive. In this study, a laboratory-built nano-flow cytometer (nFCM) that can detect single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp upon SYTO 16 staining was used to study EV-DNA at the single-vesicle level. Through simultaneous side-scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non-vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV-DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA+ ) EVs varies from 30% to 80% depending on the cell type; (3) external EV-DNA is mainly localized on relatively small size EVs (e.g. <100 nm for HCT-15 cell line) and the secretion of external DNA+ EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV-DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80-200 nm for HCT-15 cell line); (5) double-stranded DNA (dsDNA) is the predominant form of both the external and internal EV-DNA; (6) histones (H3) are not found in EVs, and EV-DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA+ EVs, and the number of both external DNA+ EVs and internal DNA+ EVs as well as the DNA content in single EVs increase significantly. This study provides direct and conclusive experimental evidence for an in-depth understanding of how DNA is associated with EVs.

Single-particle analysis of tear fluid reveals abundant presence of tissue factor-exposing extracellular vesicles with strong coagulation activity.

Extracellular vesicles (EVs) in easily accessible body fluids have emerged as a promising source for liquid biopsy. Although tear collection is fast, safe, and noninvasive, EVs of tear fluid are less studied and their involvement in physiological and pathological processes is largely unknown. The aim of present study was to analyze and characterize EVs in tear fluid at the single-particle level to reveal the population heterogeneity. A laboratory-built nano-flow cytometer (nFCM) was used to analyze the purity, size distribution, and particle concentration of EVs isolated from unstimulated tears (basal tears) upon double ultracentrifugation (17 min at 100,000×g, 4 °C) via side scattering detection. The expression of CD9, CD63, CD81, CD47, CD45, CD24, and EpCAM was assessed via immunofluorescent detection. The EV concentration in tear fluid was measured to be 1.1 ± 0.7 × 1011 particles/mL, which is approximately 100-fold higher than that of plasma EVs. In particular, it was identified for the first time that tears have strong coagulant activity owing to the abundant presence of tissue factor (TF) on tear EVs. The concentration of TF-exposing EVs (4.4 ± 3.1 × 1010 particles/mL) was found to be approximately 100-fold higher than their counterparts in saliva (4.5 ± 2.1 × 108 particles/mL). We postulate that TF-exposing vesicles in tears might play a role in host defense by promoting clot formation and thus reducing the risk of pathogen invasion. The coagulant activity of tears triggered by TF-exposing EVs could provide a new research perspective for ophthalmic research.

Sustainable production of 4-aminobutyric acid (GABA) and cultivation of Chlorella sorokiniana and Chlorella vulgaris as circular economy.

The 4-aminobutyric acid (GABA) is important to produce bio-nylon 4 in biorefineries. First, a glutamate decarboxylase (GAD) was propagated in three different Escherichia coli strains to achieve 100% conversion from 1 M monosodium glutamate after optimization of the process. To make the process greener and more efficient, in situ CO2 adaptation and citrate feeding strategies to maintain the optimal pH value and 498 g/L of GABA was obtained. However, the process releases the equivalent amount of CO2. Therefore, CO2 generated from GABA production was completely sequestered in sodium hydroxide to form bicarbonate and applied in a coupling culture of Chlorella sorokiniana (CS) or Chlorella vulgaris (CV) to increase the biomass when combined with sodium bicarbonate and carbonic anhydrase. Further improvement of 1.65-fold biomass and 1.43-fold lipid content were occurred when supplying GABA to the culture. This integrative process provided the highest GABA production rate without CO2 release, forming an eco-friendly and carbon-neutral technology.

Migration of glutamate decarboxylase by cold treatment on whole-cell biocatalyst triggered activity for 4-aminobutyric acid production in engineering Escherichia coli.

Glutamate decarboxylase B (GadB) from Escherichia coli, an intrinsic pyridoxal 5'-phosphate (PLP)-dependent enzyme has been employed for 4-aminobutyric acid (GABA) biosynthesis, which involves the glutamate import and GABA export via a transporter located in the inner membrane as rate determined step of whole-cell (WC) biotransformation. Herein, GadB was cloned and overexpressed in E. coli under a constitutive promoter in a high copy number plasmid, and 46.9 g/L GABA was produced. It was observed that GadB migrated to the periplasm when the WC were subjected to -20 °C cold treatment for 24 h prior to the biotransformation. Kinetic studies indicated that the enzymatic turnover rate of WC increased 2-fold after cold treatment, which was correlated with the migration rate of GadB, and up to 88.6% of GadB. The export or possible migration of GadB mitigated the rate-limiting step of WC biotransformation, and a 100% conversion of substrate to GABA was obtained. Finally, we launched a promising strategy for GABA production of 850 g/L from cost-effective monosodium glutamate (MSG) by using WC biocatalysts with 10-times recycling.

Experimental Study on the Mechanical Properties of Perfobond Rib Shear Connectors with Steel Fiber High Strength Concrete.

Perfobond rib (PBL) shear connectors, made up of the perforated steel plates with the penetrating rebars passing through the holes, are extensively adopted in steel-concrete composite structures for their excellent performance. The adequate understanding of mechanical properties for PBL connectors is of great significance for their reasonable design. In this study, a push out experiment, including 12 specimens with the parameters of concrete strength, diameter of penetrating rebars and the number of holes on perforated steel plate, was performed to explore the mechanical behavior of PBL connectors with steel fiber high strength concrete (SFHSC). The experimental results showed that the shear capacity of the PBL connectors increased with the increase in concrete strength, diameter of the penetrating rebars and the number of holes. Furthermore, a general prediction formula for the shear capacity of PBL connectors was developed, which considers the shear contribution of concrete dowels, concrete end-bearing, interfacial bonding between the perforated steel plates and concrete and the penetrating rebars as well as the enhancement effect of steel fibers. The prediction results of the equation are in good agreement with the experimental data and could provide a reference for the design of PBL connectors.

Experimental Investigation on the Static Performance of Stud Connectors in Steel-HSFRC Composite Beams.

In this research, high strength fiber reinforced concrete (HSFRC) was used to replace the normal strength concrete (NSC) in steel-concrete composite beams to improve their working performance, which might change the static performance of stud connectors. Firstly, push-out tests were conducted to investigation on the static performance of stud connectors in steel-HSFRC composite beams and compared with steel-NSC composite beams. Studs of 8 sizes, 13 mm, 16 mm, 19 mm and 22 mm in diameter and 80 mm and 120 mm in height were adopted to study the influence of stud dimension. The test phenomenon shown that the crack resistance of HSFRC was better than that of NSC, and there were some splitting cracks on NSC slabs whereas no visible cracks on HSFRC slabs when specimens failed. Next, the load-slip curves of studs were analyzed and a typical load-slip curve was proposed which was divided into four stages. In addition, the effects of test parameters were analyzed according to the characteristic points of load-slip curve. Compared with NSC slab, HSFRC slab could provide greater restraining force to the studs, which improved the shear capacity and stiffness of studs while suppressed the ductility of studs. The shear capacity, stiffness and ductility of studs would significantly increase with the increasement of stud diameter and the studs with large diameter were more suitable for steel-HSFRC composite beams. The stud height had no obvious influence on the static performance of studs. Finally, based on the test results, the empirical formulas for load-slip curve and shear capacity of stud connectors embedded in HSFRC were developed which considered the influence factors more comprehensively and had better accuracy and applicability than previous formulas.

Pyridoxal kinase PdxY mediated carbon dioxide assimilation to enhance the biomass in Chlamydomonas reinhardtii CC-400.

Microalga served as the promising bioresources due to the high efficiency of carbon dioxide conversion. However, the application of microalga is still restricted by low biomass, easier contamination, and high cost of production. To overcome the challenge, engineered Chlamydomonas reinhardtii CC-400 with pyridoxal kinase gene (pdxY) has demonstrated in this study. The results indicated CC-400 with pdxY reached enhanced algal biomass in three different systems, including flask, Two-layer Photo-Reactor (TPR) and airlift Photo-Bioreactor (PBR). The genetic strain PY9 cultured with 1% CO2 in the PBR showed a significant enhancement of biomass up to 1.442 g/L, a 2-times of that of the wild type. We also found the transcriptional levels of carbonic anhydrase (CA) dropped down in PY9 while higher levels of RuBisCo and pdxY occurred, thus the carbon dioxide assimilation under mixotrophic culture dramatically increased. We proofed that pdxY successfully mediated carbon dioxide utilization in CC-400.

Fabrication of bio-based polyamide 56 and antibacterial nanofiber membrane from cadaverine.

A green bioprocess for the fabrication of nanofiber membranes from the biomaterial polyamide 56 (PA56) via electrospinning was proposed. Cadaverine, as the precursor of PA56, was first produced from recombinant Escherichia coli using the whole-cell biotransformation of lysine. PA56 was then fabricated by mixing adipic acid with purified cadaverine obtained from solvent extraction and distillation. The thermal properties of the fabricated PA56 are as follows: a melting point of 250 °C, a crystallization point of 220 °C, and a degradation temperature of 410 °C. A PA56 nanofiber membrane (PAM) was further prepared via electrospinning. Dyed membranes (P-Dye) were obtained by the reaction of Reactive Red 141 dye with the amino group of PAM. Poly-(hexamethylene biguanide) (PHMB) was attached to the P-Dye to create P-Dye-PHMB. On the other hand, PAM with alginate, used to facilitate PHMB attachment (P-Alg-PHMB), was compared with P-Dye-PHMB in terms of antibacterial activity against pathogenic strains of E. coli and Pseudomonas putida. P-Alg-PHMB showed excellent antibacterial efficiency for E. coli (97%) and P. putida (100%). The proposed bioprocess can be used to fabricate novel membranes for biomedical applications and functional textiles.

Rapid quantification of pathogenic Salmonella Typhimurium and total bacteria in eggs by nano-flow cytometry.

Rapid quantification of pathogenic Salmonella Typhimurium (S. Typhimurium) and total bacteria in eggs is highly desired for food safety control. However, the complexity of egg matrix presents a significant challenge for sensitive detection of bacteria. In this study, a sample pretreatment protocol, including dilution, fat dissolution, protein degradation, filtration, and washing was developed to circumvent this challenge. A laboratory-built nano-flow cytometer (nFCM) that is hundreds of fold more sensitive than the conventional flow cytometer was employed to analyze individual bacteria upon nucleic acid and immunofluorescent staining. Eggs spiked with pathogenic S. Typhimurium and harmless Escherichia coli K12 (E. coli K12) were used as the model system to optimize the sample pretreatment protocol. S. Typhimurium and total bacteria in eggs can be quantified without cultural enrichment, and the whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1.5 h. The bacterial recovery rate upon sample pretreatment, detection limit, and dynamic range for S. Typhimurium in eggs were 92%, 2 × 103 cells/mL, and from 2 × 103 to 4 × 108 cells/mL, respectively. The as-developed approach can specifically distinguish S. Typhimurium from other bacteria and successful application to bacterial detection in eggs freshly purchased from supermarket and spoiled eggs upon inappropriate storage was demonstrated.