Dose Range-Finding Toxicity Study in Rats With Recombinant Human Lactoferrin Produced in Komagataella phaffii.

The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.

Development of Glycyrrhizinic Acid-Based Lipid Nanoparticle (LNP-GA) as An Adjuvant That Improves the Immune Response to Porcine Epidemic Diarrhea Virus Spike Recombinant Protein.

Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential -27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, which favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (~100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (p < 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1ß concentrations, which was associated with an anti-inflammatory effect.

New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Cloning, expression and molecular analysis of recombinant Netrin-A protein of Lucilia sericata Meigen (Diptera: Calliphoridae) larvae.

Objectives: Lucilia sericata (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from Lucilia sericata larvae by the baculovirus expression vector system in the Sf9 insect cell line. Methods: The coding sequence of Netrin-A was cloned, amplified in the pTG19 vector, and then cloned in the pFastBac HTA vector. It was then transformed into DH10Bac, and the recombinant Bacmid was subsequently transfected into Sf9 cells. The recombinant Netrin-A was purified by Ni-NTA agarose. The evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay. Results: The molecular weight of this protein was 52 kDa with 404 amino acids. The signal peptide was located between amino acids 24 and 25. The concentration of Netrin-A was calculated to be 48.8 µg/ml. It reaffirmed the characterized gene codes of Lucilia sericata Netrin-A in a previous study. Conclusions: The generation of recombinant Netrin-A could be used in larval therapy, and as a biomarker in certain diseases. The netrin-A of Lucilia sericata was unprecedentedly cloned and expressed in a eukaryotic cell line. Given that this larva is FDA-approved, and non-pathogenic, it conduces to research on the development of maggot therapy in future.

Removal of plant endogenous proteins from tobacco leaf extract by freeze-thaw treatment for purification of recombinant proteins.

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze-thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at - 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant ß-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Biochemical Aspects of Putative Aminotransferase Responsible for Converting Vanillin to Vanillylamine in the Capsaicinoid Biosynthesis Pathway in Capsicum Plants.

The biosynthesis pathway of capsaicinoids includes the conversion of vanillin to vanillylamine, where putative aminotransferase (pAMT) is thought to be the enzyme responsible in Capsicum plants. The objectives of this study were to prove that pAMT is the enzyme responsible for this conversion in plants and to clarify its catalytic properties using biochemical methods. Both an extract of habanero placenta and recombinant pAMT (rpAMT) constructed by using an Escherichia coli expression system were able to convert vanillin to vanillylamine in the presence of γ-aminobutyric acid as an amino donor and pyridoxal phosphate as a coenzyme. Conversion from vanillin to vanillylamine by the placenta extract was significantly attenuated by adding an anti-pAMT antibody to the reaction system. The amino donor specificity and affinity for vanillin of rpAMT were similar to those of the placenta extract. We thus confirmed that pAMT is the enzyme responsible for the conversion of vanillin to vanillylamine in capsaicinoid synthesis in Capsicum fruits. Therefore, we propose that pAMT should henceforth be named vanillin aminotransferase (VAMT).

A Novel Targeted RIG-I Receptor 5'Triphosphate Double Strain RNA-Based Adjuvant Significantly Improves the Immunogenicity of the SARS-CoV-2 Delta-Omicron Chimeric RBD-Dimer Recombinant Protein Vaccine.

The rapid mutation and spread of SARS-CoV-2 variants recently, especially through the emerging variants Omicron BA5, BF7, XBB and BQ1, necessitate the development of universal vaccines to provide broad spectrum protection against variants. For the SARS-CoV-2 universal recombinant protein vaccines, an effective approach is necessary to design broad-spectrum antigens and combine them with novel adjuvants that can induce high immunogenicity. In this study, we designed a novel targeted retinoic acid-inducible gene-I (RIG-I) receptor 5'triphosphate double strain RNA (5'PPP dsRNA)-based vaccine adjuvant (named AT149) and combined it with the SARS-CoV-2 Delta and Omicron chimeric RBD-dimer recombinant protein (D-O RBD) to immunize mice. The results showed that AT149 activated the P65 NF-κB signaling pathway, which subsequently activated the interferon signal pathway by targeting the RIG-I receptor. The D-O RBD + AT149 and D-O RBD + aluminum hydroxide adjuvant (Al) + AT149 groups showed elevated levels of neutralizing antibodies against the authentic Delta variant, and Omicron subvariants, BA1, BA5, and BF7, pseudovirus BQ1.1, and XBB compared with D-O RBD + Al and D-O RBD + Al + CpG7909/Poly (I:C) groups at 14 d after the second immunization, respectively. In addition, D-O RBD + AT149 and D-O RBD + Al + AT149 groups presented higher levels of the T-cell-secreted IFN-γ immune response. Overall, we designed a novel targeted RIG-I receptor 5'PPP dsRNA-based vaccine adjuvant to significantly improve the immunogenicity and broad spectrum of the SARS-CoV-2 recombinant protein vaccine.

pH-responsive nanocomposite hydrogel for simultaneous prevention of postoperative adhesion and tumor recurrence.

Abdominal adhesion and tumor recurrence are two thorny problems in the postoperative treatment of abdominal tumors. Although important progress has been made in the application of hydrogels in adjuvant therapy after tumor surgery, most of the products can not effectively combine the prevention of abdominal adhesion and the removal of residual cancer cells. In this study, a nanocomposite hydrogel (Col-APG-Cys@HHD) was prepared by crosslinking collagen and recombinant albumin nanoparticles (HHD NPs) with aldehydeylated polyethylene glycol (APG6K) followed by immobilizing zwitterionic cysteine (Cys) to one surface. One surface of the hydrogel adhered to the postoperative wound due to the adhesive properties of collagen, while the other surface coated with cysteine formed a hydration layer to hinder the stick of proteins and cells, thereby reducing the adhesion between tissues. Additionally, Col-APG-Cys@HHD hydrogel disintegrated under acidic condition and released HHD NPs that targeted into cancer cells and released drugs in response to low pH environment. The in vivo experiments' results demonstrated that Col-APG-Cys@HHD hydrogel could prevent intraperitoneal adhesions and inhibit tumor growth with minimal side effects, providing a potential strategy for the hydrogel-based drug delivery system in postoperative adjuvant therapy of tumors. STATEMENT OF SIGNIFICANCE: Tissue adhesion and tumor recurrence usually occur after abdominal tumor surgery. Hydrogels have been widely studied in adjuvant treatment of abdominal tumors, but their synergy in terms of controllable drug release and anti-peritoneal adhesion still needs to be improved. Herein, a nanocomposite hydrogel (Col-APG-Cys@HHD) was designed and constructed with one side that was tissue adhesive and the other side as antifouling. Additionally, the Col-APG-Cys@HHD hydrogel showed controlled drug release behavior in response to a pH gradient (6.5 to 5.5). This was conducive to its dissociation in an acidic tumor environment followed by the release of nanoparticles that entered into tumor cells and delivered docetaxel . To sum up, the Col-APG-Cys@HHD hydrogel demonstrated synergistic therapy for prevention of abdominal adhesion and tumor recurrence after abdominal tumor surgery.

Time- and cost-efficient bacterial expression and purification of potato apyrase.

Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of ∼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.

Characterization of highly gelatinous patatin storage protein from Pichia pastoris.

Patatin is a useful plant protein with excellent gelation properties that could be used as a gelling agent in the food industry. However, the commercial production of patatin is limited because the traditional extraction methods are inefficient and time consuming. Production of patatin with gelation properties by microorganisms is a promising alternative route. In this study, 1424.5 mg/L patatin storage protein with great gelation properties could be obtained in a 5-L bioreactor after optimization of the signal peptide, the promoter, and the fed-batch process when a Pichia pastoris GS115, but not Escherichia coli, expression system was used. Compared with commercial potato-extracted patatins, P. pastoris-derived patatins showed better gelation properties, such as a lower gel-forming concentration and gelation temperature. In addition, the gel strength of P. pastoris-derived patatins was comparable with that of potato-extracted patatins. These results suggested that P. pastoris-derived patatins have the potential to replace current potato-derived ones, which are now widely used in plant-based meat products.

A Novel Single-Stranded RNA-Based Adjuvant Improves the Immunogenicity of the SARS-CoV-2 Recombinant Protein Vaccine.

The research and development (R&D) of novel adjuvants is an effective measure for improving the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant protein vaccine. Toward this end, we designed a novel single-stranded RNA-based adjuvant, L2, from the SARS-CoV-2 prototype genome. L2 could initiate retinoic acid-inducible gene-I signaling pathways to effectively activate the innate immunity. ZF2001, an aluminum hydroxide (Al) adjuvanted SARS-CoV-2 recombinant receptor binding domain (RBD) subunit vaccine with emergency use authorization in China, was used for comparison. L2, with adjuvant compatibility with RBD, elevated the antibody response to a level more than that achieved with Al, CpG 7909, or poly(I:C) as adjuvants in mice. L2 plus Al with composite adjuvant compatibility with RBD markedly improved the immunogenicity of ZF2001; in particular, neutralizing antibody titers increased by about 44-fold for Omicron, and the combination also induced higher levels of antibodies than CpG 7909/poly(I:C) plus Al in mice. Moreover, L2 and L2 plus Al effectively improved the Th1 immune response, rather than the Th2 immune response. Taken together, L2, used as an adjuvant, enhanced the immune response of the SARS-CoV-2 recombinant RBD protein vaccine in mice. These findings should provide a basis for the R&D of novel RNA-based adjuvants.

[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

Determination of Histone Methyltransferase Structure by Crystallography.

As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in Escherichia coli Affects Product Yield.

Antibody fragments such as Fab's require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli, oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab's is linked to the respiratory chain, which is not only immensely important for the cell's energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab's in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.

Yeast-derived potato patatins: Biochemical and biophysical characterization.

Potato patatin is considered a valuable plant protein by the food industry for its exceptional functional properties and nutritional value. Nonetheless, it has not been widely used due to its low abundance in potatoes and high cost. Pichia pastoris was utilized for expression of patatin to overcome agricultural limitations. Biochemical and biophysical characterization of Patatin-B2 (rPatB2) and Patatin-17 (rPat17) is described. rPatB2 and rPat17 had higher zeta potential and superior solubility at various pH conditions in comparison with commercial patatin, whereas particle size distribution was similar. Inflection temperatures were higher than potato isolated patatins. Antioxidant capacity of rPatB2 and rPat17 was similar to that of commercial patatin and the specific enzymatic activity of rPatB2 was 5-fold higher than rPat17 and patatins isolated from potato. Results indicate yeast-derived patatin properties are comparable to patatins from potatoes, suggesting their potential use in various plant-based products such as meat and dairy analogues.

Effect of Recombinant Human Erythroferrone Protein on Hepcidin Gene (Hamp1) Expression in HepG2 and HuH7 Cells.

Iron is essential for all living organisms. It is strictly controlled by iron transporters, transferrin receptors, ferroportin and hepcidin. Erythroferrone (ERFE) is an iron-regulatory hormone which is highly expressed in erythroblasts by erythropoietin (EPO) stimulation and osteoblasts independently of EPO by sequestering bone morphogenetic proteins and inhibiting hepatic hepcidin expression. Although the hepcidin suppressive function of ERFE is known, its receptors still require investigation. Here, we aim to identify ERFE receptors on the HepG2 and Huh7 cells responsible for ERFE. Recombinant ERFE (rERFE) was first produced in HEK293 cells transfected with pcDNA3.1 + ERFE, then purified and detected by Western blot. The liver cells were treated with an rERFE-rich medium of transfected HEK293 cells and a purified rERFE-supplemented medium at various time points, and hepcidin gene (Hamp1) expression was determined using qRT-PCR. The results show that 37-kD rERFE was expressed in HEK293 cells. Hamp1 was suppressed at 3 h and 6 h in Huh7 cells after rERFE treatments (p < 0.05), then restored to the original levels. Hamp1 was activated after treatment with purified rERFE for 24 h and 48 h. Together, these results reveal that ERFE suppressed Hamp1 expression in liver cells, possibly acting on membrane ERFE receptor, which in Huh7 cells was more sensitive to the ERFE concentrate.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology.

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

New possible silver lining for pancreatic cancer therapy: Hydrogen sulfide and its donors.

As one of the most lethal diseases, pancreatic cancer shows a dismal overall prognosis and high resistance to most treatment modalities. Furthermore, pancreatic cancer escapes early detection during the curable period because early symptoms rarely emerge and specific markers for this disease have not been found. Although combinations of new drugs, multimodal therapies, and adjuvants prolong survival, most patients still relapse after surgery and eventually die. Consequently, the search for more effective treatments for pancreatic cancer is highly relevant and justified. As a newly re-discovered mediator of gasotransmission, hydrogen sulfide (H2S) undertakes essential functions, encompassing various signaling complexes that occupy key processes in human biology. Accumulating evidence indicates that H2S exhibits bimodal modulation of cancer development. Thus, endogenous or low levels of exogenous H2S are thought to promote cancer, whereas high doses of exogenous H2S suppress tumor proliferation. Similarly, inhibition of endogenous H2S production also suppresses tumor proliferation. Accordingly, H2S biosynthesis inhibitors and H2S supplementation (H2S donors) are two distinct strategies for the treatment of cancer. Unfortunately, modulation of endogenous H2S on pancreatic cancer has not been studied so far. However, H2S donors and their derivatives have been extensively studied as potential therapeutic agents for pancreatic cancer therapy by inhibiting cell proliferation, inducing apoptosis, arresting cell cycle, and suppressing invasion and migration through exploiting multiple signaling pathways. As far as we know, there is no review of the effects of H2S donors on pancreatic cancer. Based on these concerns, the therapeutic effects of some H2S donors and NO-H2S dual donors on pancreatic cancer were summarized in this paper. Exogenous H2S donors may be promising compounds for pancreatic cancer treatment.