Antimicrobial photodynamic therapy (APDT) is a promising alternative to traditional antibiotics for bacterial infections, which inactivates a broad spectrum of bacteria. However, it has some disadvantages including poor water solubility and easy aggregation of hydrophobic photosensitizers (PS), and poor tissue penetration and cytotoxicity when using UV as light source, leading to photodynamic therapy efficacy. Herein, we develop a novel water-soluble natural PS (sorbicillinoids) obtained by microbial fermentation using filamentous fungus Trichoderma reesei (T. reesei). Sorbicillinoids could effectively generate singlet oxygen (1O2) under UV light irradiation, and ultimately display photoinactivation activity on Gram-positive bacteria, but not Gram-negative ones. Staphylococcus aureus (S. aureus) treated with sorbicillinoids and UV light displayed high levels of intracellular reactive oxygen species (ROS), notable DNA photocleavage, and compromised membrane permeability without overt cell membrane disruption. Moreover, the dark toxicity, phototoxicity or hemolysis activity of sorbicillinoids is negligible, showing its excellent biocompatibility.
Pressure ulcers are commonly associated with microbial infections on the wounds which need an effective wound dressing. However, the silver dressings have shown promising result but they have toxicity and argyria. Hence, this study aimed to develop and characterize chitosan-polyethylene glycol (PEG) nanocomposite hydrogel loaded with phomopsidione as an antimicrobial dressing. The hydrogel being synthesized was analyzed with transmission and scanning electron microscopes. Drug release and mechanical properties were studied having confirmed the functional groups with Fourier transform infrared (FTIR) spectroscopy. Finally, antimicrobial activities were evaluated against the clinical wound pathogens. The developed hydrogel was soft, flexible and elastic, having nanospheres of chitosan-PEG but no sign of aggregation under the electron microscopes. Releasing of phomopsidione from the nanocomposite hydrogel was slow and gradual following the first order of kinetic. On average, 34 μg/mL phomopsidione released per hour and 67.9% active ingredients delivered into the surrounding medium over the study period. Although, the bioactivity activity of the hydrogel was narrow-spectrum, it showed significant results against all Gram-negative bacteria and Candida utilis with 99.99% reduction of microbial growth. The findings reveal that the phomopsidione loaded hydrogel has a great promise to act as an antimicrobial dressing for chronic wounds.
Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In the present study, expression of Vitreoscilla hemoglobin in the chromosome of Escherichia coli was used as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the fully aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the non-expressing strain. The fed-batch phase was carried out with a change of regime from aerobic to microaerobic conditions. The pDNA yields from biomass increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the non-expressing strain during microaerobic conditions. The ratio of positive/negative plasmid replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the non-expressing strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the flux in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD+ in glycolysis under microaerobic conditions. These results are valuable for faster development of robust pDNA production processes.
Urea hydrolysis is widely used in agriculture, environment and other engineering fields, among which urease contained in beans can catalyze urea hydrolysis. The urea hydrolysis activity of legume plant leaching solution(LPLS) was investigated, including soybeans, black beans, mung beans, red beans as well as soybean hulls, soybean leaves, soybean stems and soybean pods. For the high urea hydrolysis activity and economic efficiency, soybean is most suitable for agricultural engineering and other fields than other beans and soybean-related parts extract. The urea hydrolysis activity increases with the concentration of LPLS, while decreases gradually with reaction time. When the heating temperature reaches 25, 35, 45, 55 and 65 ℃, the urea hydrolysis activity is steady and the enzyme activity is high. Enzyme activity decreases after 65 ° C (i.e.75, 90 ° C). Meanwhile, the soaking time of LPLS has a little effect on the urea hydrolysis activity compared with other factors. These results make a positive contribution to domestic production urease experimental basis.
Background Application of genetically modified non-pathogenic bacteria expressing specific anti-tumor proteins under certain conditions specific to tumors is an effective approach for selective targeting of tumors. We developed here, for the first time, a novel spatiotemporal cancer targeted therapy applying engineered E. coli bacteria with capability of expressing cardiac peptides under hypoxic conditions of tumor. Method E. coli BW25133 was transformed with construction of co-expressing cardiac hormones and GFP. Bacteria bearing constructs were then IV administered in mice bearing tumors and then tumor localization, as well as tumor proliferation, invasion and angiogenesis biomarkers (Ki-67, VEGFR, CD31and MMP9), changes in cytokine profile, suppression of tumor growth and survival were analyzed. Results IV Administered bacteria bearing constructs could specifically localize at tumor site and express cardiac peptides under hypoxic conditions. Administration of bacteria significantly enhanced survival rate, suppressed tumor progression and lowered expression levels of MMP-9, VEGFR2, CD31and Ki67 as potent markers for angiogenesis, tumor proliferation and metastasis. Furthermore, applied bacteria resulted in significant reduction in the expression of IL-1β, IL-6, GC-SF, IL-12 and TNF-α proinflammatory cytokines, whereas increasing IL-10, IL-17A and INF-γ cytokines. Conclusion Overall, administration of E. coli bearing cardiac hormone expression construct could effectively suppress tumor growth, angiogenesis, invasion and metastasis while enhancing survival rate in mice model of breast cancer.
Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factors were introduction of multiple pipA genes into the whole-cell system and control of agitation. As a result, we produced 724 mM pipecolic acid (72.4% conversion), and the productivity was 0.78 g/L/h from 1 M L-lysine after 5 days. This is the highest production reported to date.
Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine crystal structures obtained to date. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for physiological function of these important drug targets. Here, we determine whether the presence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay. Upon ligand binding, the A1Δ291R or A2AΔ316R variants were unable to couple to human-yeast chimeric G-protein chimeras to generate a downstream signal in yeast, though full-length receptors showed native-like G-protein coupling. Further, constructs transfected into mammalian cells (HEK-293) showed similar behavior – i.e. the variants with C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptors. Although the C-terminus was essential for Gα protein- associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Gα (i.e. Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in signaling, but not specificity, for the interaction with Gα protein. This result has further implications in drug discovery both in enabling the experimental use of chimeras for ligand design, and in cautious interpretation of structure-based drug design based on truncated receptors.
Machine learning techniques have been successfully used to simulate and optimise bioprocesses. This study explores the feasibility to apply Gradient Boosting, an emerging Ensemble Learning algorithm, which combines weak learners to generate better predictions for bioprocess dynamic modelling and prediction. A thorough procedure was presented for Gradient Boosting based data-driven model construction. Different case studies were employed including fermentation and algal photo-production processes. Given that generating a large size of experimental data for model training is time consuming and challenging to many bioprocesses, this work launched a first investigation on the data efficiency of Gradient Boosting by comparing its predictive capability against the predominantly used artificial neural networks. By carrying out a series of experimental verifications over a broad spectrum of process operating conditions, this study concluded that Gradient Boosting may have several advantages in small experimental datasets and can outperform artificial neural networks for bioprocess predictive modelling, indicating its potential for future bioprocess digitalisation and optimisation.
Fermentation of the pentose fraction from hemicellulosic hydrolysate is an important challenge to be studied in second generation ethanol (E2G) production. In this sense, we have tested non-conventional (Scheffersomyces stipitis and Spathaspora passalidarum) and recombinant yeast strains (Saccharomyces cerevisiae recombinant MP-C5 and MP-C5H1) capable to uptake xylose with the aim to design a strategy for E2G production. Growth tests in different carbohydrates (glucose, xylose, glucose + xylose and sucrose + xylose) have shown that the integration of xylose and sucrose presented better results for all yeast tested, and the co-fermentation of these sugars provided faster xylose consumption by S. cerevisiae recombinant. One the other hand, Sp. passalidarum do not present high performance of sucrose consumption in microanaerobic fermentation conditions and an intracellular invertase high activity was observed by this yeast. S. cerevisiae MP-C5H1 presented best performance for fermentation tests and a batch strategy with high cell density was designed. By this strategy was possible to achieve high ethanol yield (0.48 g g-1), titer (53.7 g L-1) and global ethanol productivity (2.24 g L-1 h-1).
We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed: [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates lead to high purity of the recovered Abs (>95%, by gel densitometry). Good overall Ab recovery yields were observed with: Tween-20 (80-83%), Brij-O20 (85-87%) and Triton X-100 (87-90%), while Pluronic F-127 was less efficient (42-53%). Of additional importance is the finding that the process can depend on filtration (rather than centrifugation), thereby allowing a continuous purification mode that leads to the recovery of monomeric IgG’s (by DLS) and preservation of Ab specificity (by ELISA). The amphiphilic chelator, bathophenanthroline (batho) is recycled almost quantitatively (95%) by crystallization. Good IgG recovery yields (~80%) are also observed when Ab concentrations are increased from 1 mg/ml to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies (mAbs), are discussed.
his study describes the response of Arthrospira platensis to a variety of temperature conditions as reflected in variations of photosynthetic parameters, pigmentation, and biomass productivity in indoor photobioreactor (PBR) cultivations. These experiments are designed to better understand the impact of temperature, seasonal variations, and acclimation effects on outdoor biomass production. The irradiance levels and temperature range (20 – 39°C) are chosen to enable modeling of semi-continuous operation of large-scale outdoor PBR deployments. Overall, the cultivations were quite stable with some pigment-related instabilities after prolonged high temperature exposure. Changes in productivity with temperature, as reflected in measured photosynthetic parameters, are immediate and mainly attributable to the temperature dependence of the photosaturation parameter, a secondary factor being variation in pigment content. This study confirms that temperature is critical for optimal performance of Arthospira platensis for both biomass and pigment production and provides a basis for risk assessments related to temperature mitigation for large-scale outdoor cultivations. Finally, the biomass productivities in a semi-continuous operation mode are quantitatively reproduced with a productivity model incorporating the photosynthetic parameters measured herein. Those productivities are in good agreement with extensive outdoor testing in PBR arrays at large scale (24,000 L) and over long time periods (up to 18 months).
Although photodynamic therapy (PDT) is an effective, minimally-invasive therapeutic modality with advantages in highly localized and specific tumor treatments, large and deep-seated cancers within the body cannot be successfully treated due to low transparency to visible light. To improve the therapeutic efficiency of tumor treatment in deep tissue and reduce the side effects in normal tissue, this study developed a near-infrared (NIR)-triggered upconversion nanoparticle (UCNP)-based photosensitizer (PS) carrier as a novel theranostics system. The NaYF4:Yb/Er UCNPs were synthesized by a hydrothermal method, producing uniformly small size (≈20 nm) nanoparticles and crystalline morphology of the hexagonal phase. These UCNPs were modified with folic acid-conjugated biocompatible block copolymers through a bidentate dihydrolipoic acid linker. The polymer modified hexagonal phase UCNPs (FA-PEAH-UCNPs) showed an improved dispersibility in the aqueous solution and strong NIR-to-vis upconversion fluorescence. The hydrophobic PS, pheophorbide a (Pha), was then conjugated to the stable vectors through a pH-sensitive linkage. Moreover, these UCNP-based Pha carriers containing tumor targeting folic acid ligands exhibited the significantly enhanced cellular uptake efficiency as well as PDT treatment efficiency. These results suggested that this system could extend the excitation wavelength of PDT to the NIR region and effectively improve therapeutic efficiency of PSs.