Contralesional Application of Transcranial Direct Current Stimulation on Functional Improvement in Ischemic Stroke Mice.

BACKGROUND AND PURPOSE: The therapeutic use of transcranial direct current stimulation (tDCS), an adjuvant tool for stroke, induces long-term changes in cortical excitability, for example, the secretion of activity-dependent growth factors. We assessed the proper therapeutic configuration of high-definition tDCS (HD-tDCS) in the subacute stage of ischemic stroke and its underlying expression profiling of growth factors to propose a new method for ensuring better therapeutic effects. METHODS: Male C57BL/6J mice were subjected to middle cerebral artery occlusion, after which repetitive HD-tDCS (20 minutes, 55 µA/mm2, charge density 66 000 C/m2) was applied from subacute phases of their ischemic insult. Behavioral tests assessing motor and cognitive functions were used to determine suitable conditions and HD-tDCS stimulation sites. Gene expression profiling of growth factors and their secretion and activation were analyzed to shed light on the underlying mechanisms. RESULTS: Anodal HD-tDCS application over the contralesional cortex, especially the motor cortex, was more effective than ipsilesional stimulation in attenuating motor and cognitive deficits. In the HD-tDCS application over the contralesional motor cortex, positive changes in Bmp8b, Gdf5, Il4, Pdgfa, Pgf, and Vegfb were observed in the ipsilesional site. The expression of GDF5 (growth/differentiation factor 5) and PDGFA (platelet-derived growth factor subunit A) tended to similarly increase in both ipsi- and contralesional striata. However, higher expression levels of GDF5 and PDGFA and their receptors were observed in the peri-infarct regions of the striatum after HD-tDCS, especially in PDGFA expression. A higher number of proliferating or newly formed neuronal cells was detected in ipsilesional sites such as the subventricular zone. CONCLUSIONS: Application of anodal HD-tDCS over the contralesional cortex may enhance beneficial recovery through the expression of growth factors, such as GDF5 and PDGFA, in the ipsilesional site. Therefore, this therapeutic configuration may be applied in the subacute stage of ischemic stroke to ameliorate neurological impairments.

Transcriptome Analysis of the Striatum of Electroacupuncture-treated Naïve and Ischemic Stroke Mice.

Objectives: Electroacupuncture (EA) has been demonstrated to aid stroke recovery. However, few investigations have focused on identifying the potent molecular targets of EA by comparing EA stimulation between naïve and disease models. Therefore, this study was undertaken to identify the potent molecular therapeutic mechanisms underlying EA stimulation in ischemic stroke through a comparison of mRNA sequencing data obtained from EA-treated naïve control and ischemic stroke mouse models. Methods: Using both naïve control and middle cerebral artery occlusion (MCAO) mouse models, EA stimulation was administered at two acupoints, Baihui (GV20) and Dazhui (GV14), at a frequency of 2 Hz. Comprehensive assessments were conducted, including behavioral evaluations, RNA sequencing to identify differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction (PPI) network analysis, and quantitative real-time PCR. Results: EA stimulation ameliorated the ischemic insult-induced motor dysfunction in mice with ischemic stroke. Comparative analysis between control vs. MCAO, control vs. control + EA, and MCAO vs. MCAO + EA revealed 4,407, 101, and 82 DEGs, respectively. Of these, 30, 7, and 1 were common across the respective groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed upregulated DEGs associated with the regulation of inflammatory immune response in the MCAO vs. MCAO + EA comparison. Conversely, downregulated DEGs in the control vs. control + EA comparison were linked to neuronal development. PPI analysis revealed major clustering related to the regulation of cytokines, such as Cxcl9, Pcp2, Ccl11, and Cxcl13, in the common DEGs of MCAO vs. MCAO + EA, with Esp8l1 identified as the only common downregulated DEG in both EA-treated naïve and ischemic models. Conclusion: These findings underscore the diverse potent mechanisms of EA stimulation between naïve and ischemic stroke mice, albeit with few overlaps. However, the potent mechanisms underlying EA treatment in ischemic stroke models were associated with the regulation of inflammatory processes involving cytokines.

Therapeutic effects of a novel electrode for transcranial direct current stimulation in ischemic stroke mice.

Rationale: Non-invasive transcranial direct current stimulation (tDCS), a promising stimulation tool to modulate a wide range of brain disorders, has major limitations, such as poor cortical stimulation intensity and focality. We designed a novel electrode for tDCS by conjugating a needle to a conventional ring-based high-definition (HD) electrode to enhance cortical stimulation efficacy. Method: HD-tDCS (43 µA/mm2, charge density 51.6 kC/m2, 20 min) was administered to male C57BL/6J mice subjected to early-stage ischemic stroke. Behavioral tests were employed to determine the therapeutic effects, and the underlying mechanisms of HD-tDCS were determined by performing RNA sequencing and other biomedical analyses. Results: The new HD-tDCS application, showing a higher electric potential and spatial focality based on computational modeling, demonstrated better therapeutic effects than conventional HD-tDCS in alleviating motor and cognitive deficits, with a decrease in infarct volume and inflammatory response. We assessed different electrode configurations in the new HD electrode; the configurations variously showed potent therapeutic effects, ameliorating neuronal death in the peri-infarct region via N-methyl-D-aspartate-dependent sterol regulatory element-binding protein 1 signaling and related inflammatory factors, further alleviating motor and cognitive deficits in stroke. Conclusion: This new HD-tDCS application showed better therapeutic effects than those with conventional HD-tDCS in early-stage stroke via the amelioration of neuronal death in the penumbra. It may be applied in the early stages of stroke to alleviate neurological impairment.

Validating a Xylose Regulator to Increase Polyhydroxybutyrate Production for Utilizing Mixed Sugars from Lignocellulosic Biomass Using Escherichia coli.

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is economically beneficial. Because lignocellulosic biomass is a mixture rich in glucose and xylose, Escherichia coli, which prefers glucose, needs to overcome glucose repression for efficient biosugar use. To avoid glucose repression, here, we overexpressed a xylose regulator (xylR) in an E. coli strain expressing bktB, phaB, and phaC from Cupriavidus necator and evaluated the effect of xylR on PHB production. XylR overexpression increased xylose consumption from 0% to 46.53% and produced 4.45-fold more PHB than the control strain without xylR in a 1% sugar mixture of glucose and xylose (1:1). When the xylR-overexpressed strain was applied to sugars from lignocellulosic biomass, cell growth and PHB production of the strain showed a 4.7-fold increase from the control strain, yielding 2.58 ± 0.02 g/l PHB and 4.43 ± 0.28 g/l dry cell weight in a 1% hydrolysate mixture. XylR overexpression increased the expression of xylose operon genes by up to 1.7-fold. Moreover, the effect of xylR was substantially different in various E. coli strains. Overall, the results showed the effect of xylR overexpression on PHB production in a non-native PHB producer and the possible application of xylR for xylose utilization in E. coli.

Sex-specific responses to juvenile stress on the dopaminergic system in an animal model of attention-deficit hyperactivity disorder.

The etiology of attention-deficit hyperactivity disorder (ADHD) strongly suggests a genetic component as the main cause; however, environmental factors such as early adverse experiences in childhood may play an interactive role with the genetic susceptibility. Spontaneously hypertensive rats (SHRs), a genetic ADHD model, and control Wistar Kyoto rats (WKYs) were subjected to chronic unpredictable mild stress during the juvenile period. The behavioral characteristics were monitored, and dopamine-related factors in the core regions of dopaminergic pathways were measured. Higher ADHD symptom-related behaviors were observed in response to juvenile stress in male SHRs than control WKYs. For the SHRs subjected to juvenile stress, hyperactivity in males, recognition in females, and depressant potential in both sexes were markedly observed. In the expression of 17 dopamine-related genes and proteins, greater changes were detected in male SHRs subjected to juvenile stress, especially in dopamine metabolic factors. Dopamine clearance factors involved in dopamine degradation and transport, especially catechol-O-methyltransferase (COMT) and dopamine transporter (DAT), showed sex-specific differences induced by juvenile stress in dopamine metabolite assays. Moreover, stressed male SHRs treated with methylphenidate showed better improvement in behavior than the females, resulting in different levels of COMT and DAT amelioration. These results suggest that juvenile stress potentially increased the incidence of ADHD in a genetic rat model, which showed sex-specific differences based on the expression of COMT and DAT. Therefore, our results could help develop gender-specific diagnostics and healthcare options for juvenile stress in patients with ADHD.

Sialocele and Its Association with Hypercortisolism and Long-Term Glucocorticoid Treatment in Dogs: Retrospective Case-Control Study.

Dogs with sialocele often have concurrent hypercortisolism or are receiving long-term glucocorticoid treatment. However, their association has not been investigated. This retrospective matched case-control study investigated the association between hypercortisolism, long-term glucocorticoid treatment, and sialocele in dogs. We retrospectively reviewed the records from 1 January 2018 to 31 December 2022. Records of 19 dogs diagnosed with sialocele were investigated for hypercortisolism and long-term glucocorticoid treatment. Two age- and breed-matched controls for each sialocele dog (38 dogs) were investigated for the same concurrent diseases. Logistic regression analysis was used. The odds of sialocele in dogs with hypercortisolism were 15.56 times those of dogs without hypercortisolism (p = 0.02; 95% CI: 1.54-156.79). The odds of sialocele in dogs with long-term glucocorticoid treatment (median, 8 months; range, 5-13) were 7.78 times those of dogs without long-term glucocorticoid treatment (p = 0.03; 95% CI: 1.23-49.40). No associations were found between age, sex, body weight, and the presence of sialocele. The results indicate that sialocele was significantly associated with hypercortisolism and long-term glucocorticoid treatment in dogs. Therefore, dogs with hypercortisolism or receiving long-term glucocorticoid therapy should be screened for possible sialocele. Additionally, dogs with sialocele should be identified for concurrent hypercortisolism and prolonged glucocorticoid exposure.

Enhancement of polyhydroxybutyrate production by introduction of heterologous phasin combination in Escherichia coli.

Phasin is a surface-binding protein of polyhydroxyalkanoate (PHA) granules that is encoded by the phaP gene. As its expression increases, PHA granules become smaller, to increase their surface area, and are densely packed inside the cell, thereby increasing the PHA content. A wide range of PHA-producing bacteria have phaP genes; however, their PHA productivity differs, although they are derived from the cognate bacterial host cell. Modulating phasin expression could be a new strategy to enhance PHA production. This study aimed to characterize the effect of heterologous phasins on the reconstitution of E. coli BL21(DE3) and determine the best synergistic phaP gene combination to produce polyhydroxybutyrate (PHB). We identified novel phasins from a PHB high-producer strain, Halomonas sp. YLGW01, and introduced a combination of phaP genes into Escherichia coli. The resulting E. coli phaP1,3 strain had enhanced PHB production by 2.9-fold, leading to increased cell mass and increased PHB content from 48 % to 65 %. This strain also showed increased tolerance to inhibitors, such as furfural and vanillin, enabling the utilization of lignocellulose biosugar as a carbon source. These results suggested that the combination of phaP1 and phaP3 genes from H. sp. YLGW01 could increase PHB production and robustness.

Transcranial alternating current stimulation rescues motor deficits in a mouse model of Parkinson's disease via the production of glial cell line-derived neurotrophic factor.

BACKGROUND: Therapeutic effects of transcranial alternating current stimulation (tACS) for treating Parkinson's disease (PD) are limited to modulating abnormally synchronized oscillations; however, long-lasting tACS effects may involve non-neuronal mechanisms like the regulation of neurotrophic factors. OBJECTIVES/HYPOTHESIS: We investigated whether tACS exerts neuroprotective effects on dopaminergic neurons in a mouse model of PD by regulating endogenous glial cell line-derived neurotrophic factor (GDNF). METHODS: Repeated high-definition tACS (HD-tACS, 20 min, 89.1 µA/mm2) was administered over the primary motor cortex of C57BL/6J 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. Behavioral tests assessing motor function, immunohistochemistry, western blots, enzyme-linked immunosorbent assays, and flow cytometric analyses were performed to examine suitable tACS conditions and its underlying mechanisms. RESULTS: Stimulation at representative frequencies (theta to gamma; 20-Hz beta frequency, in particular) attenuated motor dysfunction and protected the dopaminergic neurons with increased GDNF production. Beta-frequency (20 Hz) tACS application significantly attenuated motor deficits to levels comparable with those of levodopa treatment. Moreover, beta-frequency tACS induced the survival of dopaminergic neurons in the substantia nigra with upregulated production of endogenous GDNF in striatal parvalbumin-positive interneurons. An inhibitor of the GDNF receptor-associated rearranged during transfection (RET) kinase suppressed most aspects of the tACS-induced behavioral recovery, dopaminergic cell survival, and GDNF production. Beta-frequency tACS activated RET-related survival signaling for dopaminergic neurons in the substantia nigra. CONCLUSIONS: Application of tACS over the primary motor cortex may exert protective effects on dopaminergic neurons in the substantia nigra via activation of endogenous GDNF production by striatal parvalbumin-positive interneurons and its survival signaling.

Effects of electroacupuncture on the functionality of NG2-expressing cells in perilesional brain tissue of mice following ischemic stroke.

Neural/glial antigen 2 (NG2)-expressing cells has multipotent stem cell activity under cerebral ischemia. Our study examined the effects of electroacupuncture (EA) therapy (2 Hz, 1 or 3 mA, 20 minutes) at the Sishencong acupoint on motor function after ischemic insult in the brain by investigating the rehabilitative potential of NG2-derived cells in a mouse model of ischemic stroke. EA stimulation alleviated motor deficits caused by ischemic stroke, and 1 mA EA stimulation was more efficacious than 3 mA EA stimulation or positive control treatment with edaravone, a free radical scavenger. The properties of NG2-expressing cells were altered with 1 mA EA stimulation, enhancing their survival in perilesional brain tissue via reduction of tumor necrosis factor alpha expression. EA stimulation robustly activated signaling pathways related to proliferation and survival of NG2-expressing cells and increased the expression of neurotrophic factors such as brain-derived neurotrophic factor, tumor growth factor beta, and neurotrophin 3. In the perilesional striatum, EA stimulation greatly increased the number of NG2-expressing cells double-positive for oligodendrocyte, endothelial cell, and microglia/macrophage markers (CC1, CD31, and CD68). EA therapy also greatly activated brain-derived neurotrophic factor/tropomyosin receptor kinase B and glycogen synthase kinase 3 beta signaling. Our results indicate that EA therapy may prevent functional loss at the perilesional site by enhancing survival and differentiation of NG2-expressing cells via the activation of brain-derived neurotrophic factor -induced signaling, subsequently ameliorating motor dysfunction. The animal experiments were approved by the Animal Ethics Committee of Pusan National University (approval Nos. PNU2019-2199 and PNU2019-2884) on April 8, 2019 and June 19, 2019.

Finding of novel lactate utilizing Bacillus sp. YHY22 and its evaluation for polyhydroxybutyrate (PHB) production.

Polyhydroxyalkanoates (PHAs) and their derivatives are biopolymers that have the potential of replacing petroleum-based plastics and can be produced and degraded via bacterial metabolism. However, there are only a few studies on polyhydroxybutyrate (PHB) production using lactate, one of the major waste organic acids that could be implemented in the production of polylactic acid (PLA). Herein, we screened and characterized the PHA-producing microbial strains isolated from saltern soil from Docho Island (South Korea). Among the 24 identified microorganisms that can use lactate as a carbon source, Bacillus sp. YHY22, a newly reported strain, produced the highest amount of PHB: 4.05 g/L with 6.25 g/L dry cell weight, which is 64.7% PHB content under optimal production conditions. Bacillus sp. YHY22 could form the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer with propionate addition. Moreover, Bacillus sp. YHY22 produced PHB in non-sterilized 2% lactate and 8% NaCl marine broth culture medium, suggesting that its production can occur in high salinity media without additional sterilization steps, rendering fermentation cost- and time-efficient.

Controlling catabolite repression for isobutanol production using glucose and xylose by overexpressing the xylose regulator.

Using lignocellulosic biomass is immensely beneficial for the economical production of biochemicals. However, utilizing mixed sugars from lignocellulosic biomass is challenging because of bacterial preference for specific sugar such as glucose. Although previous studies have attempted to overcome this challenge, no studies have been reported on isobutanol production from mixed sugars in the Escherichia coli strain. To overcome catabolite repression of xylose and produce isobutanol using mixed sugars, we applied the combination of three strategies: (1) deletion of the gene for the glucose-specific transporter of the phosphotransferase system (ptsG); (2) overexpression of glucose kinase (glk) and glucose facilitator protein (glf); and (3) overexpression of the xylose regulator (xylR). xylR gene overexpression resulted in 100% of glucose and 82.5% of xylose consumption in the glucose-xylose mixture (1:1). Moreover, isobutanol production increased by 192% in the 1:1 medium, equivalent to the amount of isobutanol produced using only glucose. These results indicate the effectiveness of xylR overexpression in isobutanol production. Our findings demonstrated various strategies to overcome catabolite repression for a specific product, isobutanol. The present study suggests that the selected strategy in E. coli could overcome the major challenge using lignocellulosic biomass to produce isobutanol.

Study of SarA by DNA Affinity Capture Assay (DACA) Employing Three Promoters of Key Virulence and Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most well-known human pathogens, houses many virulence factors and regulatory proteins that confer resistance to diverse antibiotics. Although they have been investigated intensively, the correlations among virulence factors, regulatory proteins and antibiotic resistance are still elusive. We aimed to identify the most significant global MRSA regulator by concurrently analyzing protein-binding and several promoters under same conditions and at the same time point. DNA affinity capture assay (DACA) was performed with the promoters of mecA, sarA, and sarR, all of which significantly impact survival of MRSA. Here, we show that SarA protein binds to all three promoters. Consistent with the previous reports, ΔsarA mutant exhibited weakened antibiotic resistance to oxacillin and reduced biofilm formation. Additionally, production and activity of many virulence factors such as phenol-soluble modulins (PSM), α-hemolysin, motility, staphyloxanthin, and other related proteins were decreased. Comparing the sequence of SarA with that of clinical strains of various lineages showed that all sequences were highly conserved, in contrast to that observed for AgrA, another major regulator of virulence and resistance in MRSA. We have demonstrated that SarA regulates antibiotic resistance and the expression of various virulence factors. Our results warrant that SarA could be a leading target for developing therapeutic agents against MRSA infections.

Leucyl-tRNA Synthetase Inhibitor, D-Norvaline, in Combination with Oxacillin, Is Effective against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe diseases in humans. For decades, MRSA has acquired substantial resistance against conventional antibiotics through regulatory adaptation, thereby posing a challenge for treating MRSA infection. One of the emerging strategies to combat MRSA is the combinatory use of antibacterial agents. Based on the dramatic change in phospholipid fatty acid (PLFA) composition of MRSA in previous results, this study investigated branched-chain amino acid derivatives (precursors of fatty acid synthesis of cell membrane) and discovered the antimicrobial potency of D-norvaline. The compound, which can act synergistically with oxacillin, is among the three leucine-tRNA synthetase inhibitors with high potency to inhibit MRSA cell growth and biofilm formation. PLFA analysis and membrane properties revealed that D-norvaline decreased the overall amount of PLFA, increasing the fluidity and decreasing the hydrophobicity of the bacterial cell membrane. Additionally, we observed genetic differences to explore the response to D-norvaline. Furthermore, deletion mutants and clinically isolated MRSA strains were treated with D-norvaline. The study revealed that D-norvaline, with low concentrations of oxacillin, was effective in killing several MRSA strains. In summary, our findings provide a new combination of aminoacyl-tRNA synthetase inhibitor D-norvaline and oxacillin, which is effective against MRSA.

Novel Polyhydroxybutyrate-Degrading Activity of the Microbulbifer Genus as Confirmed by Microbulbifer sp. SOL03 from the Marine Environment.

Ever since bioplastics were globally introduced to a wide range of industries, the disposal of used products made with bioplastics has become an issue inseparable from their application. Unlike petroleum-based plastics, bioplastics can be completely decomposed into water and carbon dioxide by microorganisms in a relatively short time, which is an advantage. However, there is little information on the specific degraders and accelerating factors for biodegradation. To elucidate a new strain for biodegrading poly-3-hydroxybutyrate (PHB), we screened out one PHB-degrading bacterium, Microbulbifer sp. SOL03, which is the first reported strain from the Microbulbifer genus to show PHB degradation activity, although Microbulbifer species are known to be complex carbohydrate degraders found in high-salt environments. In this study, we evaluated its biodegradability using solid- and liquid-based methods in addition to examining the changes in physical properties throughout the biodegradation process. Furthermore, we established the optimal conditions for biodegradation with respect to temperature, salt concentration, and additional carbon and nitrogen sources; accordingly, a temperature of 37°C with the addition of 3% NaCl without additional carbon sources, was determined to be optimal. In summary, we found that Microbulbifer sp. SOL03 showed a PHB degradation yield of almost 97% after 10 days. To the best of our knowledge, this is the first study to investigate the potent bioplastic degradation activity of Microbulbifer sp., and we believe that it can contribute to the development of bioplastics from application to disposal.

Adsorptive removal of synthetic plastic components bisphenol-A and solvent black-3 dye from single and binary solutions using pristine pinecone biochar.

The existing study deals with adsorptive removal of the endocrine-disrupting chemical bisphenol-A and toxic azo dye solvent black-3 from single and binary solutions. These two chemicals are commonly used as an additive in the synthetic plastic industries. Among the tested twenty pristine and modified biochars, the pristine pinecone biochar produced at 750 °C revealed greater bisphenol-A removal. Simulation of the experimental data obtained for bisphenol-A and dye removal from the single-component solution offered a best-fit to Elovich (R2 > 0.98) and pseudo-second-order (R2 > 0.99) kinetic models, respectively. Whereas for the bisphenol-A + dye removal from binary solution, the values for bisphenol-A adsorption were best suited to Elovich (R2 > 0.98), while pseudo-second-order (R2 > 0.99) for dye removal. Similarly, the two-compartment model also demonstrated better values (R2 > 0.92) for bisphenol-A and dye removal from single and binary solutions with greater Ffast values (except for bisphenol-A in binary solution). The Langmuir isotherm model demonstrated the highest regression coefficient values (R2 > 0.99) for bisphenol-A and dye removal with the highest adsorption capacity of 38.387 mg g-1 and 346.856 mg g-1, correspondingly. Besides, the co-existence of humic acid revealed a positive impact on bisphenol-A removal, while the dye removal rate was slightly hindered in presence of humic acid. The absorption process showed monolayer coverage of biochar surface with contaminants using a chemisorption mechanism with fast reactions between functional groups on the adsorbate and adsorbent. Whereas the adsorption mechanism was primarily controlled by hydrogen bonding, hydrophobic and π-π electron-donor-acceptor interactions as confirmed by FTIR, XPS, and pH investigations.

Polyhydroxyalkanoates (PHAs) degradation by the newly isolated marine Bacillus sp. JY14.

Polyhydroxyalkanoates (PHAs) are bioplastic substitutes for petroleum-derived plastics that may help to reduce the increasing environmental impact of plastic pollution. Among them, polyhydroxybutyrate (PHB) is a promising biopolymer, incentivizing many researchers to search for PHB-producing and PHB-degrading bacteria for improved PHB utilization. Many novel PHB-producing microorganisms have been discovered; however, relatively few PHB-degrading bacteria have been identified. Six PHB-degrading bacteria were found in marine soil and investigated their PHB-degrading abilities under various temperature and salinity conditions using solid-media based culture. Finally, thermotolerant and halotolerant PHB-degrader Bacillus sp. JY14 was selected. PHB degradation was confirmed by monitoring changes in the physical and chemical properties of PHB films incubated with Bacillus sp. JY14 using scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. Further, PHB degradation ability of Bacillus sp. JY14 was measured in liquid culture by gas chromatography. After 14 days of cultivation with PHB film, Bacillus sp. JY14 achieved approximately 98% PHB degradation. Applying various bioplastics to assess the bacteria's biodegradation capabilities, the result showed that Bacillus sp. JY14 could also degrade P(3HB-co-4HB) and P(3HB-co-3HV). Overall, this study identified a thermotolerant and halotolerant bacteria capable of PHB degradation under solid and liquid conditions. These results suggest that this bacteria could be utilized to degrade various PHAs.

Screening of the strictly xylose-utilizing Bacillus sp. SM01 for polyhydroxybutyrate and its co-culture with Cupriavidus necator NCIMB 11599 for enhanced production of PHB.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.

Novel phasins from the Arctic Pseudomonas sp. B14-6 enhance the production of polyhydroxybutyrate and increase inhibitor tolerance.

Phasin (PhaP), one of the polyhydroxyalkanoate granule-associated protein, enhances cell growth and polyhydroxybutyrate (PHB) biosynthesis by regulating the number and size of PHB granules. However, few studies have applied phasins to various PHB production conditions. In this study, we identified novel phasin genes from the genomic data of Arctic soil bacterium Pseudomonas sp. B14-6 and determined the role of phaP1Ps under different PHB production conditions. Transmission electron microscopy and gel permeation chromatography revealed small PHB granules with high-molecular weight, while differential scanning calorimetry showed that the extracted PHB films had similar thermal properties. The phasin protein derived from Pseudomonas sp. B14-6 revealed higher PHB production and exhibited higher tolerance to several lignocellulosic biosugar-based inhibitors than the phasin protein of Ralstonia eutropha H16 in a recombinant Escherichia coli strain. The increased tolerance to propionate, temperature, and other inhibitors was attributed to the introduction of phaP1Ps, which increased PHB production from lignocellulosic hydrolysate (2.39-fold) in the phaP1Ps strain. However, a combination of phasin proteins isolated from two different sources did not increase PHB production. These findings suggest that phasin could serve as a powerful means to increase robustness and PHB production in heterologous strains.

Comparative Study of the Difference in Behavior of the Accessory Gene Regulator (Agr) in USA300 and USA400 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA).

Community-associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is notorious as a leading cause of soft tissue infections. Despite several studies on the Agr regulator, the mechanisms of action of Agr on the virulence factors in different strains are still unknown. To reveal the role of Agr in different CA-MRSA, we investigated the LACΔagr mutant and the MW2Δagr mutant by comparing LAC (USA300), MW2 (USA400), and Δagr mutants. The changes of Δagr mutants in sensitivity to oxacillin and several virulence factors such as biofilm formation, pigmentation, motility, and membrane properties were monitored. LACΔagr and MW2Δagr mutants showed different oxacillin sensitivity and biofilm formation compared to the LAC and MW2 strains. Regardless of the strain, the motility was reduced in Δagr mutants. And there was an increase in the long chain fatty acid in phospholipid fatty acid composition of Δagr mutants. Other properties such as biofilm formation, pigmentation, motility, and membrane properties were different in both Δagr mutants. The Agr regulator may have a common role like the control of motility and straindependent roles such as antibiotic resistance, biofilm formation, change of membrane, and pigment production. It does not seem easy to control all MRSA by targeting the Agr regulator only as it showed strain-dependent behaviors.

Engineering of Shewanella marisflavi BBL25 for biomass-based polyhydroxybutyrate production and evaluation of its performance in electricity production.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.