Self-cleaved expression of recombinant lysostaphin from its cellulose binding domain fusion.

Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst. KEY POINTS: • CBD fusion enhanced soluble expression of recombinant lysostaphin. • In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions. • Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.

Nanofibrous Membrane with Encapsulated Glucose Oxidase for Self-Sustained Antimicrobial Applications.

Polyvinyl alcohol (PVA) nanofibrous membrane, consisting of separately encapsulated glucose oxidase (GOx) and glucose (Glu) nanofibers, was prepared via simultaneously electrospinning PVA/GOx and PVA/Glu dopes. The as-prepared pristine membrane could self-sustainably generate hydrogen peroxide (H2O2) only in contact with an aqueous solution. The H2O2 production level was well maintained even after storing the dry membrane at room temperature for 7 days. Cross-linking the membrane via reaction with glutaraldehyde (GA) vapor could not only prevent the nanofibrous membrane from dissolving in water but also prolonged the release of H2O2. The sustained release of H2O2 from the membrane achieved antimicrobial capability equivalent to that of 1% H2O2 against both Escherichia coli and Staphylococcus aureus. Gram(+) S. aureus cells were more susceptible to H2O2 than Gram(-) E. coli and >99% of S. aureus were killed after 1 h incubation with the membrane. Pristine and GA-crosslinked nanofibrous membrane with in situ production of H2O2 were self-sterilized in which no microorganism contamination on the membrane could be detected after 2 weeks incubation on an agar plate. The GOx/Glu membrane may find potential application as versatile antimicrobial materials in the field of biomedicine, in the food and health industries, and especially challenges related to wound healing in diabetic patients.

Class I hydrophobin fusion with cellulose binding domain for its soluble expression and facile purification.

Hydrophobins, highly surface-active proteins, have the ability to reverse surface hydrophobicity through self-assembly at the hydrophilic-hydrophobic interfaces. Their unique structure and interfacial activity lead hydrophobins to have potential applications on surface functional modifications. However, class I hydrophobins are prone to self-assemble into highly insoluble amyloid-like rodlets structure. Recombinant hydrophobins could be produced by Escherichia coli but generally as an insoluble inclusion body. To overcome this insoluble expression limitation, cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of class I hydrophobin HGFI to enhance its soluble expression in E. coli. Approximately, 94% of expressed CBD fused HGFI (CBD-HGFI) was found as soluble protein. The fused CBD could also bind specifically onto bacterial cellulose (BC) nanofibrils produced by Komagataeibacter xylinus to facilitate rapid isolation and purification of HGFI from crude extract. Lysostaphin (Lst), known as GlyGly endopeptidase could successfully cleave the flexible linker (GGGGS)2 between CBD and HGFI to recover HGFI from BC-bound CBD-HGFI. CBD-HGFI purified by immobilized metal-chelated affinity chromatography (IMAC) and Lst cleaved BC-CBD-HGFI still retained interfacial activity of hydrophobin and its effect on accelerating PETase hydrolysis against poly(ethylene terephthalate) (PET) fiber.

Recent Advances in Novel Lateral Flow Technologies for Detection of COVID-19.

The development of reliable and robust diagnostic tests is one of the most efficient methods to limit the spread of coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, most laboratory diagnostics for COVID-19, such as enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR), are expensive, time-consuming, and require highly trained professional operators. On the other hand, the lateral flow immunoassay (LFIA) is a simpler, cheaper device that can be operated by unskilled personnel easily. Unfortunately, the current technique has some limitations, mainly inaccuracy in detection. This review article aims to highlight recent advances in novel lateral flow technologies for detecting SARS-CoV-2 as well as innovative approaches to achieve highly sensitive and specific point-of-care testing. Lastly, we discuss future perspectives on how smartphones and Artificial Intelligence (AI) can be integrated to revolutionize disease detection as well as disease control and surveillance.

Efficient Design to Monitor the Site-specific Sustained Release of a Non-Emissive Anticancer Drug.

A pH-responsive smart nanocarrier with significant components was synthesized by conjugating the non-emissive anticancer drug methyl orange and polyethylene glycol derived folate moiety to the backbone of polynorbornene. Complete synthesis procedure and characterization methods of three monomers included in the work: norbornene-derived Chlorambucil (Monomer 1), norbornene grafted with polyethylene glycol, and folic acid (Monomer 2) and norbornene attached methyl orange (Monomer 3) connected to the norbornene backbone through ester linkage were clearly discussed. Finally, the random copolymer CHO PEG FOL METH was synthesized by ring-opening metathesis polymerization (ROMP) using Grubbs' second-generation catalyst. Advanced polymer chromatography (APC) was used to find the final polymer's molecular weight and polydispersity index (PDI). Dynamic light scattering, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were utilized to explore the prodrug's size and morphology. Release experiments of the anticancer drug, Chlorambucil and the coloring agent, methyl orange, were performed at different pH and time. Cell viability assay was carried out for determining the rate of survived cells, followed by the treatment of our final polymer named CHO PEG FOL METH.

Class I hydrophobins pretreatment stimulates PETase for monomers recycling of waste PETs.

Poly(ethylene terephthalate) hydrolase (PETase) from Ideonella sakaiensis 201-F6 was expressed and purified from Escherichia coli to hydrolyze poly(ethylene terephthalate) (PET) fibers waste for its monomers recycling. Hydrolysis carried out at pH 8 and 30 °C was found to be the optimal condition based on measured monomer mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) concentrations after 24 h reaction. The intermediate product bis(2-hydroxyethyl) terephthalate (BHET) was a good substrate for PETase because BHET released from PET hydrolysis was efficiently converted into MHET. Only a trace amount of MHET could be further hydrolyzed to TPA. Class I hydrophobins RolA from Aspergillus oryzae and HGFI from Grifola frondosa were expressed and purified from E. coli to pretreat PET surface for accelerating PETase hydrolysis against PET. The weight loss of hydrolyzed PET increased from approximately 18% to 34% after hydrophobins pretreatment. The releases of TPA and MHET from HGFI-pretreated PET were enhanced 48% and 62%, respectively. The selectivity (TPA/MHET ratio) of the hydrolysis reaction was approximately 0.5.

The green synthesis of a palm empty fruit bunch-derived sulfonated carbon acid catalyst and its performance for cassava peel starch hydrolysis.

A sulfonated carbon acid catalyst (C-SO3H) was successfully generated from palm empty fruit bunch (PEFB) carbon via hydrothermal sulfonation via the addition of hydroxyethylsulfonic acid and citric acid. The C-SO3H catalyst was identified as containing 1.75 mmol g-1 of acid and 40.2% sulphur. The surface morphology of C-SO3H shows pores on its surface and the crystalline index (CrI) of PEFB was decreased to 63.8% due to the change structure as it became carbon. The surface area of the carbon was increased significantly from 11.5 to 239.65 m2 g-1 after sulfonation via hydrothermal treatment. The identification of -SO3H, COOH and -OH functional groups was achieved using Fourier-transform infrared spectroscopy. The optimal catalytic activity of C-SO3H was achieved via hydrolysis reaction with a yield of 60.4% of total reducing sugar (TRS) using concentrations of 5% (w/v) of both C-SO3H and cassava peel starch at 100 °C for 1 h. The stability of C-SO3H shows good performance over five repeated uses, making it a good potential candidate as a green and sulfonated solid acid catalyst for use in a wide range of applications.

Fungal Hydrophobin RolA Enhanced PETase Hydrolysis of Polyethylene Terephthalate.

Polyethylene terephthalate (PET) becomes one of the most well-known polyesters and is widely used as packaging material. Recently, polyethylene terephthalate hydrolase (PETase) has emerged as a potential biocatalyst demonstrating the ability to degrade polyethylene terephthalate (PET). We showed that the rate of PETase hydrolysis could be significantly increased in the presence of hydrophobin RolA. Hydrophobins represent a class of small fungal protein that has a high surface-active substance and can spontaneously self-assemble at hydrophilic-hydrophobic interfaces. In this work, a class I hydrophobin named RolA was extracted from the mycelium pellet collected from a fermentation culture of Aspergillus oryzae. The SDS-PAGE analysis of the isolated RolA showed the presence of 11 kDa polypeptide. Recombinant PETase from Ideonella sakaiensis was also successfully expressed in Escherichia coli as a soluble protein with molecular weight approximately 30 kDa. The hydrophobin RolA could enhance the PET hydrolysis in the presence of the recombinant PETase. The hydrolysis of PET bottle by RolA-PETase achieved the highest weight loss of 26% in 4 days. It is speculated that the wetting effect of RolA acts on PET surface converts PET to become hydrophilic that leads PETase easier to contact and attack the surface. Graphical Abstract.

Hyaluronic Acid Derived Hypoxia-Sensitive Nanocarrier for Tumor Targeted Drug Delivery.

Hyaluronic acid (HA) is conjugated with BHQ3 moiety with azo bonds to prepare hypoxia-responsive polymer conjugate. Because of the amphiphilic nature, the polymer conjugate self-assembles to HA-BHQ3 nanoparticles (NPs). The anticancer drug doxorubicin (DOX) is loaded into the NPs. In the physiological environment, DOX is released slowly. In contrast, under hypoxic conditions, the azo bond in BHQ3 is cleaved, thus significantly enhancing the DOX release rate. For instance, after 24 h, 25% of DOX is released under normal conditions, while 74% of DOX is released under hypoxic conditions. In vitro cytotoxicity demonstrates higher toxicity in the hypoxic conditions. DOX@HA-BHQ3 NPs exhibit greater toxicity levels against 4T1 cells in hypoxic conditions. The fluorescent microscope images confirm the oxygen-dependent intracellular DOX release from the NPs. The in vivo biodistribution results suggest the tumor targetability of HA-BHQ3 NPs in 4T1 tumor-bearing mice.

Surface Functionalization of Poly(N-Vinylpyrrolidone) onto Poly(Dimethylsiloxane) for Anti-Biofilm Application.

Poly(dimethylsiloxane) (PDMS) has been widely used in the field of microfluidics, optical systems, and sensors. However, the hydrophobic nature of PDMS leads to low surface wettability and biofouling problems due to the nonspecific proteins-hydrophobic surface interactions and cell/bacterial adhesion. In this work, the PDMS surface was first introduced with amino groups (PDMS-NH2) via KOH-catalyzed reaction with 3-aminopropyltriethoxysilane (APTES). The PDMS-NH2 was then grafted with poly(N-vinylpyrrolidone) (PVP) based on the self-adhesion reaction between the amino surface and catechol-functionalized PVP (CA-PLL-PVP). CA-PLL-PVP as a comb-polymer was synthesized by conjugating PVP-COOH along with caffeic acid to the ε-polylysine backbone. A significantly enhanced water wettability was observed with contact angles dropped from 116° to 14° after coating with CA-PLL-PVP. The coated surface demonstrated excellent antifouling performance that no appreciable Staphylococcus epidermidis biofilm formation could be observed. This novel facile antifouling coating on PDMS surface may find greater biomedical applications to eliminate the potential adherence problems caused by natural biofouling.

Cellulose binding domain fusion enhanced soluble expression of fructosyl peptide oxidase and its simultaneous purification and immobilization.

Hemoglobin A1c (HbA1c) is a hemoglobin molecule in which the N-terminal valine residue of the ß subunit has been grafted with the glucose in blood. Its detection has important implications for the diagnosis of diabetes. Enzymatic colorimetric method using fructosyl peptide oxidase (FPO) is simple and rapid for HbA1c detection. A FPO mutant with enhanced activity was constructed and produced by E. coli; however, most of expressed mutant FPO was insoluble. Significantly enhanced expression solubility was achieved when cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of FPO mutant. Via the high affinity interaction between CBD and cellulose, the CBD fusion also facilitated the simultaneous purification and immobilization of FPO directly from E. coli cells lysate using bacterial cellulose (BC) nanofibrils as a matrix of very high specific area. A never-dried and water durable nanocellulose film with FPO activity could be easily obtained by collecting the FPO immobilized BC nanofibrils suspension on the surface of a microfiltration membrane. The activity of the ready-use FPO nanocellulose film was stable at least 7 days at room temperature for the detection of HbA1c level of 5.3-11% in blood samples.

Recombinant fructosyl peptide oxidase preparation and its immobilization on polydopamine coating for colorimetric determination of HbA1c.

Recombinant fructosyl peptide oxidase (FPO) from Phaeosphaeria nodorum SN15 was functionally expressed by Escherichia coli cells and one-step purified from crude cells extract using immobilized metal affinity chromatography (IMAC) to achieve a specific activity of 26 U/mg. A ready-use colorimetric detection of HbA1c level in blood sample was developed based on FPO immobilized membrane. Facile bio-inspired polydopamine coating on the surface of a microporous membrane was employed for effective FPO immobilization. Glutaraldehyde activation of the polydopamine coating significantly enhanced FPO immobilization yield that at least 5-fold higher activity could be achieved. The stability of FPO membrane was also enhanced by glutaraldehyde activation that 85% activity could be maintained after 7 repeated uses. Highly correlated optical densities at 727 nm (OD727) against fructosylvaline (FV) in the range of 0.02 mM to 0.7 mM (R2 = 0.988) could be achieved using FPO membrane. At least 80% of the initial activity was maintained for FPO membrane stored at 4 °C for 7 days. Rather low OD727 but good correlation still could be obtained by using FPO membrane for the detection HbA1c levels (6-14%) in blood samples.

Twofold enhanced dispersin B activity by N-terminal fusion to silver-binding peptide for biofilm eradication.

Dispersin B (DspB) has shown a great potential for the hydrolysis of polymeric ß-1,6-N-acetyl-d-glucosamine (PNAG) to disperse the biofilms formed by various bacteria but with no killing activity. Here we have investigated whether a silver-binding peptide (AgBP) fused to DspB can induce the in situ formation of silver nanoparticles (AgNP) and conjugated to the structure of DspB so that the bacteria cells released from the dispersed biofilm will be killed by the conjugated AgNP. However, the desired conjugate could be obtained because of the silver ions itself was found to precipitate DspB. But, the fusion of AgBP2 to DspB (AgBP2-DspB) could generate at least 2 fold higher activity against soluble substrate 4-nitrophenyl N-acetyl-ß-D-glucosaminide (NP-GlcNAc). By applying to a preformed Staphylococcus epidermidis biofilm, AgBP2-DspB could clear 69% of the biofilm while only 37% could be cleared by DspB as observed by fluorescent microscope. As measured by crystal violet staining, biofilm could be eradicated to the same extent by loading AgBP2-DspB activity level approximately 20 fold lower than that of DspB. The biofilm formation could be prevented on a AgBP2-DspB immobilized surface as observed by confocal laser microscope.

Antimicrobial sponge prepared by hydrophobically modified chitosan for bacteria removal.

Hydrophobically modified chitosan (HMCS) prepared by reacting chitosan with dodecyl aldehyde can generate very stable foam when dissolved in mild acidic condition under vigorous mechanical stirring. A durable and lightweight (density of 32 mg/ml) sponge was obtained by freeze-drying the stably formed HMCS foam. In addition to the cationic nature of chitosan, the grafted C12 alkyl chains were also able to help HMCS sponge for capturing E. coli cells (∼4.0 × 108 cells/mg sponge) by intercalating into the outer membrane of E. coli cells. E. coli cells captured on HMCS sponge were found to be mostly dead and easily released into the bulk solution so that the active surface could be continuously regenerated for capturing and killing the rest of alive cells. In comparison with its counterpart (chitosan sponge), HMCS sponge maintained a higher operational stability for the removal of E. coli cells. After 5 repeated cells removal operation, the removal capacity of HMCS sponge could be regenerated back to >90% by thorough washing with ethanol.

Pt-MWCNT modified carbon electrode strip for rapid and quantitative detection of H2O2 in food.

A single-use screen-printed carbon electrode strip was designed and fabricated. Nanohybrids, prepared by deposition of platinum (Pt) nanoparticles on multi-wall carbon nanotube (MWCNT), was modified on the surface of screen-printed carbon electrode for the development of a fast, sensitive and cost-effective hydrogen peroxide (H2O2) detection amperometric sensor strip. With Pt-MWCNT nanohybrids surface modification, current generated in response to H2O2 by the screen-printed carbon electrode strip was enhanced 100 fold with an applied potential of 300 mV. Quality of as-prepared electrode strip was assured by the low coefficient of variation (CV) (<5%) of currents measured at 5 s. Three linear detection ranges with sensitivity of 75.2, 120.7, and 142.8 µA mM-1 cm-2 were observed for H2O2 concentration in the range of 1-15 mM, 0.1-1 mM, and 10-100 µM, respectively. The lowest H2O2 concentration could be measured by the as-prepared strip was 10 µM. H2O2 levels in green tea infusion and pressed Tofu could be rapidly detected with results comparable to that measured by ferrous oxidation xylenol orange (FOX) assay and peroxidase colorimetric method.

Iodine-loaded metal organic framework as growth-triggered antimicrobial agent.

Zeolitic imidazolate framework-8 (ZIF-8) is one of easily available metal organic frameworks because of its facile preparation via mixing aqueous solutions of zinc nitrate and 2-methylimidazole. It turned into a very effective pH-responsive bactericide after loading with iodine. Approximately, 0.9g of iodine could be readily loaded into one gram of ZIF-8 from iodine dissolved n-heptane solution. Both Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis and Staphylococcus aureus could be very effectively killed by iodine loaded ZIF-8 (ZIF-8@I) at pH6.0 within 3min. In contrast, at pH above 7.0, no appreciable antimicrobial activity could be detected. The bacteria killing effect is resulted from the iodine released from ZIF-8@I disintegrated at acidic pH. ZIF-8@I coated surface also showed its acidic pH-triggered antimicrobial activity against deposited bacterial cells. The antimicrobial activity of ZIF-8@I against actively grown bacterial lawns on a pH neutral agar plate was also observed. The result demonstrates that iodine was released from the disintegrated ZIF-8@I to kill bacteria in response to the bacterial growth-induced pH lowering.

Cells capture and antimicrobial effect of hydrophobically modified chitosan coating on Escherichia coli.

Hydrophobically modified chitosan (HMCS), prepared by reacting alkyl aldehyde with chitosan was demonstrated to be an effective antimicrobial and transparent coating. The grafted alkyl chains exist as protruded hydrophobic tails on the HMCS coating surface. In contact with E. coli cells, HMCS coating captured and immobilized the cells via these hydrophobic tails. The hydrophobic tails could also kill the cells captured on the coating surface as visualized by fluorescence microscope. More than 50% of the initially loaded cells (2.5×104 CFU) could be killed after 2h contact with HMCS coating. The cells capture and killing effects of the coating surface could be completely neutralized by treating with α-cyclodextrin to sequester the protruded hydrophobic tails. The facile coating of antimicrobial HMCS on surface also enabled the easy fabrication of patterned E. coli cells arrays.

Silver deposited carboxymethyl chitosan-grafted magnetic nanoparticles as dual action deliverable antimicrobial materials.

Carboxymethyl chitosan (CMCS) was known to have a much better antimicrobial activity than chitosan due to the increased cationic -NH3+ groups resulted from the intra- and intermolecular interactions between the carboxyl and amino groups. CMCS was grafted onto the surface of silica coated magnetic nanoparticles (MNPs) to obtain magnetically retrievable and deliverable antimicrobial nanoparticles (MNPs@CMCS). The presence of carboxylate groups in CMCS not only enhanced antimicrobial activity but also enabled Ag ions chelating ability to induce the in situ formation of Ag nanoparticles (AgNPs). The deposition of AgNPs on the surface of MNPs@CMCS could significantly increase its antimicrobial activity against planktonic cells due to the dual action of CMCS and AgNPs. Due to its high magnetism, the as-prepared MNPs@CMCS-Ag could be efficiently delivered into an existing biofilm under the guidance of an applied magnetic field. Without direct contact, the Ag ions and/or radical oxygen species (ROS) released from the deposited Ag nanoparticles could effectively kill the bacteria embedded in the extracellular polymeric substances (EPS) matrix of biofilm.

Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris.

PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6 × His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P. pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P. pastoris transformant. Highest hyaluronidase activity of 2 mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230 mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100-300 kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4 h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC50) of 8 mM against rHuPH20.

Bactericidal magnetic nanoparticles with iodine loaded on surface grafted poly(N-vinylpyrrolidone).

Bactericidal magnetic nanoparticles were prepared by complexing iodine with poly(N-vinylpyrrolidone) (PVP) grown at the surface of silica coated magnetic nanoparticles (MNPs) via surface-initiated atom transfer radical polymerization (SI-ATRP). Approximately, 10 mg of iodine could be loaded onto one gram of the PVP-grafted MNPs to form bactericidal MNPs@PVP-I. At a concentration of 5 g L-1, MNPs@PVP-I could achieve 100% bactericidal rate for both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus with a concentration of ∼1 × 1010 CFU mL-1 within 3 min. After being used for killing the bacteria in solution, the bactericidal rate of the MNPs@PVP-I decreased to <10% due to the consumption of iodine. The bactericidal rate could be tuned back to 100% when the used MNPs@PVP-I was recharged in a 15 g L-1 iodine solution for 12 h. The as-prepared bactericidal MNPs@PVP-I could be reused at least 4 times with 100% bactericidal rate by repeatedly recharging it with iodine.