Green alternatives to petroleum-based plastics: production of bioplastic from Pseudomonas neustonica strain NGB15 using waste carbon source.

Polyhydroxyalkanoates have attracted great interest as a suitable alternative to petrochemical based plastics due to their outstanding properties such as biodegradability and biocompatibility. However, the biggest problem in the production of microbial polyhydroxyalkanoates is low cost-effectiveness. In this study, polyhydroxyalkanoate production was carried out using waste substrates with local isolates. Culture conditions were optimized to increase the polyhydroxyalkanoate production potential. The produced polyhydroxyalkanoate was characterized by FTIR analyses, and its metabolic pathway was determined by real-time PCR. According to the results, the best polyhydroxyalkanoate producer bacteria was characterized as Pseudomonas neustonica NGB15. The optimal culture conditions were detected as 30 g/L banana peel powder, 25 °C temperature, pH 8, and 4-day incubation time. Under the optimized conditions, 3.34 g/L PHA production was achieved. As a result of FTIR analyses, major peaks were obtained at 1723, 1277, 1261, 1097, 1054, and 993 cm-1. These peaks represent that the type of produced polyhydroxyalkanoate was poly-ß-hydroxybutyrate. According to gene expression profile of NGB15, it was determined that Pseudomonas neustonica NGB15 produces PHA using the de novo fatty acid synthesis metabolic pathway. In conclusion, poly-ß-hydroxybutyrate production by Pseudomonas neustonica NGB15 using a low-cost fermentation medium has been shown to be biotechnologically promising.

Expression of Human ß-defensin 2 (hBD-2) in Pichia Pastoris and Investigation of Its Binding Efficiency with ACE-2.

COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the mild and severe transition of COVID-19. Antimicrobial peptides (AMPs), which are important components of the innate immune system, are potential molecules to fight pathogenic bacteria, fungi, and viruses. Human ß-defensin 2 (hBD-2), a 41-amino-acid antimicrobial peptide, is one of the defensins inducibly expressed in the skin, lungs, and trachea in humans. In this study, it was aimed to investigate the interaction of hBD-2 produced recombinantly in Pichia pastoris with the human angiotensin-converting enzyme 2 (ACE-2) under in vitro conditions. First, hBD-2 was cloned in P. pastoris X-33 via the pPICZαA vector, a yeast expression platform, and its expression was confirmed by SDS-PAGE, western blotting, and qRT-PCR. Then, the interaction between recombinant hBD-2 and ACE-2 proteins was revealed by a pull-down assay. In light of these preliminary experiments, we suggest that the recombinantly produced hBD-2 may be protective against SARS-CoV-2 and be used as a supplement in treatment. However, current findings need to be supported by cell culture studies, toxicity analyses, and in vivo experiments.

A new hydrophobin candidate from Cladosporium macrocarpum with super-hydrophobic surface.

Hydrophobins are amphipathic proteins with small molecular weights produced in filamentous fungi. These proteins are highly stable due to the disulfide bonds formed between the protected cysteine residues. They have great potential for usage in many different fields such as surface modifications, tissue engineering, and drug transport systems because hydrophobins are surfactants and soluble in harsh mediums. In this study, it was aimed to determine the hydrophobin proteins responsible for the hydrophobicity of the super-hydrophobic fungi isolates in the culture medium and to carry out the molecular characterization of the hydrophobin producer species. As a result of measuring surface hydrophobicity by determining the water contact angle, five different fungi with the highest hydrophobicity were classified as Cladosporium by classical and molecular (ITS and D1-D2 regions) methods. Also, protein extraction according to the recommended method for obtaining hydrophobins from spores of these Cladosporium species indicated that the isolates have similar protein profiles. Ultimately, the isolate named A5 with the highest water contact angle was identified as Cladosporium macrocarpum, and the 7 kDa band was appointed as a hydrophobin since it was the most abundant protein in protein extraction for this species.

Evaluation of Nisin-Loaded PLGA Nanoparticles Prepared with Rhamnolipid Cosurfactant against S. aureus Biofilms.

In this article, nisin(N)-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) were prepared using the single-solvent evaporation method with a rhamnolipid(R) cosurfactant. The antibacterial-antibiofilm effects of the prepared formulation and free nisin were evaluated against S. aureus (ATCC 25923). The characterization of NPs was analyzed using scanning electron microscopy (SEM), Zetasizer and Fourier-transform infrared spectroscopy (FTIR). The drug encapsulation efficiency and loading capacity percentages of NPs were calculated by the spectrophotometric method. The drug release of N-loaded PVA-R-PLGA NPs was determined by the dialysis bag method. The antibacterial and antibiofilm activity of N-PVA-R-PLGA NPs was determined. PVA-R-PLGA-NPs were found to be spherical with sizes of ~140 nm, according to the SEM analysis and surface charge of N-PVA-R-PLGA NPs -53.23 ± 0.42 mV. The sustained release of N (≥72% after 6 h) was measured in PVA-R-PLGA-NPs. The encapsulation efficiency percentage of N-PVA-R-PLGA NP was 78%. The MIC values of free nisin and N-PVA-R-PLGA NPs were 256 µg/mL and 64 µg/mL, respectively. The antibiofilm inhibition percentages of free nisin and N-PVA-R-PLGA NPs were 28% and 72%, respectively. These results reveal that N-PVA-R-PLGA NPs are a promising formulation for use in infections caused by S. aureus compared to free nisin.

MicroRNA-145 transcriptionally regulates Semaphorin 3A expression in prostate cancer cells.

Prostate cancer (PCa) is one of the most prevalent cancer types among males. Differential expression of microRNAs is associated with various cancers including PCa. Although mature microRNAs are preferentially located in the cytoplasm, several studies identified mature human microRNAs in purified nuclei and miR-145 has been found to be predominantly expressed in the nuclei of benign tissues compared to tumor lesions. However, the nuclear functions of miR-145 are yet limited. Here, we aimed at investigating the inductive role of miR-145 on the expression of Semaphorin 3A (SEMA3A) in PCa cell lines. To study the regulatory potential of miR-145 in the transcriptional level in PCa, we overexpressed miR-145 in PC3 and DU145 cells, and confirmed its upregulation by quantitative-real-time-PCR. Then we investigated the tumor suppressor potential of miR-145 upon inducing SEMA3A expression using cell viability assay, western blot analysis, Chromatin Immunoprecipitation assay and luciferase reporter assay. Our results revealed that p53, miR-145, and SEMA3A expressions are significantly downregulated in PC3 and DU145 cells compared to nontumorigenic prostate epithelial PNT1a cells. miR-145 overexpression in PCa cells induced the expression of SEMA3A at both messenger RNA and protein levels. Furthermore, increased miR-145 expression enriched RNA Pol-II antibody on the promoter of SEMA3A and induced luciferase activity controlled by SEMA3A promoter. In this study, we showed that the functions of miR-145 are not limited to gene silencing, and found that it may lead to changes in gene expression in the transcriptional level.

Screening and characterization of a novel Antibiofilm polypeptide derived from filamentous Fungi.

In the present study, 120 fungal isolates were locally isolated from soil and selected according to their ability to antimicrobial activity. Then, selected isolates were tested for their ability to prevent biofilm formation and only one isolate (A01) showed an antibiofilm effect. The isolate A01 identified as Aspergillus tubingensis by sequencing of the 18S ITS region and a segment of ß-tubulin gene. Then, 5 fractions were prepared from the culture filtrate of A. tubingensis A01 using the ultrafiltration technique to find active polypeptide fraction. The experiments revealed that one of them had an antibiofilm activity. The MALDI-TOF/MS analyses demonstrated that this polypeptide composed of 92 amino acids and had a molecular mass of 10,087 Da. The sequence alignment showed homology with hypothetical protein (OJI81679.1). The gene coding for this polypeptide consisting of 279 nucleotides, herein we called astucin, was cloned and sequenced from A. tubingensis A01 to confirm results. The MIC of the purified polypeptide was 32 m/L and 128 µg/mL and the MBIC was 2 and 8 µg/mL against Staphylococcus aureus and MRSA, respectively. The results demonstrated that the antimicrobial and antibiofilm activity of astucin, together with its lack of cytotoxicity, makes it an alternative for application in medicine. SIGNIFICANCE: Antibiotic resistance is a global problem and the emergence of antibiotic resistant bacteria reduce the effect the current treatment approaches. In this context, antimicrobial peptides stand out as potentional agents to combat bacterial infection especially, biofilm related infections. Importantly, this study have greatly considered our understanding for fungal derived antibiofilm polypeptides. In this study, traditional selection method combined with crystal violet assay is used to investigate antibiofilm polypeptides. We identified antibiofilm polypeptides purified from A. tubingensis A01. This protein shows antimicrobial and antibiofilm activity against S. aureus.

ING5 inhibits cancer aggressiveness by inhibiting Akt and activating p53 in prostate cancer.

Prostate cancer (PCa) is one of the most common types of cancer in men. In several recent studies, chromosomal deletions in the q arm of chromosome 2, where ING5 resides within, have been identified in various cancer types including PCa. In this study, we investigate the role of ING5 as a tumor suppressor in PCa. We examined the expression level of ING5 in tissue samples and cell lines using quantitative real-time polymerase chain reaction and western blot analysis. We tested the in vitro tumor suppressor potential of ING5 in PC3 and LNCaP cells stably overexpressing it using cell viability, colony formation, migration, invasion, and apoptosis assays. We then investigated the effects of ING5 on the Akt and p53 signaling using western blot analysis. We show that ING5 is significantly downregulated in PCa tumor tissue samples and cell lines compared with the corresponding controls. In vitro assays demonstrate that ING5 effectively suppresses proliferative, clonogenic, migratory, and invasive potential and induce apoptosis in PCa cells. ING5 may potentially exert its anti-tumor potential by inhibiting AKT and inducing p53 signaling pathways. Our findings demonstrate that ING5 possesses tumor suppressor roles in vitro, pointing its importance during the prostatic carcinogenesis processes.

Carvacrol prodrugs as novel antimicrobial agents.

Carvacrol (CAR), a natural monoterpene particularly abundant in plants belonging to the Lamiaceae family, has recently attracted much attention for its many biological properties (antioxidant, anti-inflammatory, neuroprotective, antitumour, antibacterial, and several others). However, CAR has poor chemical-physical properties (low water solubility and high volatility), which hamper its potential pharmacological uses. In this paper, the synthesis and antimicrobial evaluation of 23 carvacrol derivatives (WSCP1-23) against a panel of selected gram-positive and gram-negative bacteria are reported. Using the prodrug approach, CAR hydrophilic (WSCP1-17) and lipophilic prodrugs (WSCP18-23) were prepared. Notably, CAR water solubility was increased by using polar neutral groups (such as natural amino acids) with the aim of improving oral drug delivery. On the other hand, CAR lipophilic prodrugs, obtained by prenylation of CAR hydroxyl group, were designed to promote membrane permeation and oral absorption. Our results revealed that WSCP1-3, showing the highest water solubility (>1700-fold compared to that of CAR), possessed good antibacterial activity against gram-negative bacteria with MIC values comparable to those of CAR and antifungal properties against different species of Candida. WSCP18-19 were the most promising prodrugs, showing good antibacterial profiles against gram-positive bacteria by interfering with the biofilm formation of Staphylococcus aureus and Staphylococcus epidermidis. Moreover, WSCP18-19 resulted more stable in simulated fluids and human plasma than WSCP1-3. Toxicity studies performed on human erythrocytes and HaCaT cells revealed that all WSCPs were not toxic at the tested concentrations.

Natural-based Antibiofilm and Antimicrobial Peptides from Microorganisms.

As the resistance to antimicrobial molecules increases among bacteria, the need for new antimicrobial molecules increases. Antimicrobial peptides (AMP), which may be a new generation of antibiotic candidates, are important in this respect. AMPs are small, cationic and amphipathic peptide sequences. In eukaryotes, they are synthesized as a part of the immune system. Substantially, AMPs are discovered in all kingdoms of life such as bacteria, fungi and protozoa. Approximately 3,000 AMPs have been reported in the literature. However, most of these AMPs have been synthesized through chemical synthesis. Nature has a huge source of microorganisms, and in the literature, there is a tendency to increase every year the number of bacteria and fungus-derived AMPs thanks to their biotechnological importance. The exploration of AMP and antibiofilm peptide (ABP) producer microorganisms brings with it a lot of challenges experimentally. In this review study, we want to highlight the importance and challenge of these natural peptides derived from microorganisms. We will also propose a new explanation for ABPs.

Chicken feather peptone: A new alternative nitrogen source for pigment production by Monascus purpureus.

Peptones are accepted as one of the most favourable nitrogen sources supporting pigment synthesis in Monascus purpureus. The present study was performed to test the feasibility of chicken feather peptone (CFP) as nitrogen source for pigment production from M. purpureus ATCC16365. CFP was compared with fish peptone (FP) and protease peptone (PP) in order to elucidate its effectiveness on pigment production. CFP was prepared from waste feathers using hydrolysis (KOH) and neutralization (H2SO4) methods. The protein content of CFP was determined as 67.2 g/100 g. Optimal concentrations of CFP and glucose for pigment production were determined as 3 and 20 g/L, respectively. A medium pH of 5.5 and an incubation period of 7-days were found to be more favourable for pigment production. In CFP, PP and FP media, yellow pigment absorbances were 2.819, 2.870 and 2.831, red pigment absorbances were 2.709, 2.304 and 2.748, and orange pigment absorbances were 2.643, 2.132 and 2.743, respectively. Sugar consumption and mycelia growth showed the similar trends in CFP, FP and PP media. This study indicates that the peptone from chicken feathers may be a good nutritional substrate for pigment production from M. purpureus.

A Laboratory Assessment of Two Local Strains of the Beauveria bassiana (Bals.) Vuill. against the Tetranychus urticae (Acari: Tetranychidae) and Their Potential as a Mycopesticide.

This study was conducted to assess highly pathogenic Beauveria bassiana isolates to be used in biocontrol and to determine their potentials as mycopesticide. For this purpose, two B. bassiana isolates, which were locally isolated from T. urticae, were chosen. Firstly, three suspensions were investigated at the degree of humidity of 65 ± 5% and 100% RH. Secondly, these strains were selected according to their tendency to mass production, tolerance to UV radiation, and capability of producing spore at the different temperatures. Finally, identification of the selected isolate was performed by using ITS rDNA analysis. Both tested fungal isolates were pathogenic to the T. urticae. Mycelial growths of isolate AT076 at 20°C and 30°C were found to be greater than isolate AT007. It was observed that isolate AT076 had more spore production with 1.61 × 107 spore/disc at 30°C and 44.33% germination after UV radiation for 15 min. The numbers of spores per 5 mm disk area for isolates AT076 and AT007 were found to be 1.2 × 106 and 1.0 × 106. These results show that isolate AT076 was more virulent and more UV-tolerant and had higher tendency to mass production compared to isolate AT007 against T. urticae. As a result of this study, isolate AT076 can be used in the biocontrol as mycopesticide.

Nitric oxide: a novel inducer for enhancement of microbial lipase production.

The purpose of this study was to elucidate whether exogenous nitric oxide (NO) has a potential beneficial effect on lipase production capacity of some microorganisms. Sodium nitroprusside (SNP) was used as an exogenous NO donor in production medium. In comparison with the control (0 nM SNP), SNP concentrations from 10 to 100 nM induced lipase production in mesophilic bacterium Bacillus subtilis and cold-adapted yeast Yarrowia lipolytica. Especially, the maximum lipase activities for Y. lipolytica (81.2 U/L) and B. subtilis (74.5 U/L) were attained at 30 and 50 nM SNP concentrations, respectively. When compared to the control, the optimal SNP concentrations resulted in about 5.14 and 2.27-fold increases in lipase activities of B. subtilis and Y. lipolytica, respectively. Besides, it was found that the optimal SNP concentrations provided shorter incubation periods for lipase production. Conversely, no significant positive effect of exogenous NO on lipase production was determined for thermophilic bacterium Geobacillus stearothermophilus. This study showed for the first time that exogenous NO could be used as an inducer in the production of microbial lipases.

Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

Enhancement of invertase production by Aspergillus niger OZ-3 using low-intensity static magnetic fields.

The aim of this study is to investigate the effect of low-intensity static magnetic fields (SMFs) on invertase activity and growth on different newly identified molds. The most positive effect of SMFs on invertase activity and growth was observed for Aspergillus niger OZ-3. The submerged production of invertase was performed with the spores obtained at the different exposure times (120, 144, 168, and 196 hr) and magnetic field intensities (0.45, 3, 5, 7, and 9 mT). The normal magnetic field of the laboratory was assayed as 0.45 mT (control). Optimization of magnetic field intensity and exposure time significantly increased biomass production and invertase activity compared to 0.45 mT. The maximum invertase activity (51.14 U/mL) and biomass concentration (4.36 g/L) were achieved with the spores obtained at the 144 hr exposure time and 5 mT magnetic field intensity. The effect of low-intensity static magnetic fields (SMFs) on invertase activities of molds was investigated for the first time in the present study. As an additional contribution, a new hyper-invertase-producing mold strain was isolated.