Carbon recycling with synthetic CO2 fixation pathways.

Carbon dioxide (CO2) is the node of alleviating global climate change and supporting living organisms on Earth. Currently, the warming climate and the growing population demand enhanced CO2 fixation for a sustainable future, which stimulates innovations in biotechnology to tackle these challenges. To this endeavor, synthetic biology and metabolic engineering are enabling a promising approach to engineer synthetic carbon fixation in heterotrophic organisms combining the advantages of both autotrophs and heterotrophs. Here, we review the current advances in constructing synthetic CO2 fixation pathways and discuss the underlying design principles with confronting challenges. Moreover, we highlight the application scenarios of these designs at different concentrations of CO2, and how sustainable bioproduction can be improved. We also foresee the future of engineering synthetic carbon fixation pathways for carbon recycling.

Cellular differentiation into hyphae and spores in halophilic archaea.

Several groups of bacteria have complex life cycles involving cellular differentiation and multicellular structures. For example, actinobacteria of the genus Streptomyces form multicellular vegetative hyphae, aerial hyphae, and spores. However, similar life cycles have not yet been described for archaea. Here, we show that several haloarchaea of the family Halobacteriaceae display a life cycle resembling that of Streptomyces bacteria. Strain YIM 93972 (isolated from a salt marsh) undergoes cellular differentiation into mycelia and spores. Other closely related strains are also able to form mycelia, and comparative genomic analyses point to gene signatures (apparent gain or loss of certain genes) that are shared by members of this clade within the Halobacteriaceae. Genomic, transcriptomic and proteomic analyses of non-differentiating mutants suggest that a Cdc48-family ATPase might be involved in cellular differentiation in strain YIM 93972. Additionally, a gene encoding a putative oligopeptide transporter from YIM 93972 can restore the ability to form hyphae in a Streptomyces coelicolor mutant that carries a deletion in a homologous gene cluster (bldKA-bldKE), suggesting functional equivalence. We propose strain YIM 93972 as representative of a new species in a new genus within the family Halobacteriaceae, for which the name Actinoarchaeum halophilum gen. nov., sp. nov. is herewith proposed. Our demonstration of a complex life cycle in a group of haloarchaea adds a new dimension to our understanding of the biological diversity and environmental adaptation of archaea.

CO2 bubble-assisted in-situ construction of mesoporous Co-doped Cu2(OH)2CO3 nanosheets as advanced electrodes towards fast and highly efficient electrochemical reduction of nitrate to N2 in wastewater.

The development of high-efficient and cost-effective electrocatalysts is crucial to remove nitrate pollutant in wastewater. Herein, we design and prepare mesoporous Co-doped Cu2(OH)2CO3 malachite nanosheets as an electrocatalyst toward highly efficient nitrate reduction using a facile CO2 bubble-assisted coprecipitation synthesis. The electrocatalytic performance is subject to the Co/Cu ratio of this malachite. Remarkably, compared with the pristine monometal Cu or Co-based electrocatalyst, the optimal electrocatalyst, 0.3Co@Cu2(OH)2CO3, displays fast and highly efficient removal capacity of nitrate with an impressive high total nitrogen (TN) removal of 8628.99 mg N g-1CoCu (398.79 mg N gcat-1 h-1), N2 selectivity of 97.11% as well as negligible nitrite product at 100 mg L-1 NO3--N and 2000 mg L-1 Cl- neutral electrolyte. Above all, high total nitrogen removal efficiency (81.92%) and chemical oxygen demand (73.74%) in actual wastewater. Its excellent electrocatalytic performance is achieved by regulating the electronic structure and the adsorption/desorption of the intermediate. This study discovers a new type of electrode materials for nitrate removal in wastewater.

Glycine receptors expression in rat spinal cord and dorsal root ganglion in prostaglandin E2 intrathecal injection models.

BACKGROUND: Glycine receptors (GlyRs) are involved in the development of spinal pain sensitization. The GlyRα3 subunit has recently emerged as a key factor in inflammatory pain pathways in the spinal cord dorsal horn (DH). Our study is to identify the extent of location and cell types expressing different GlyR subunits in spinal cord and dorsal root ganglion (DRGs). To tease out the possible actions of GlyRs on pain transmission, we investigate the effects produced by GlyRs on acute inflammatory pain by behavioral testing using prostaglandin E2 (PGE2) intrathecal injection models. Furthermore, we investigate the changes of GlyR expression in DRGs and spinal cord in rats after the induction of acute inflammatory pain. RESULTS: Compared to the vehicle administration, the PGE2 intrathecal injection model produced significantly higher hyperalgesia, which started 3 h after PGE2 injection and lasted more than 5 h. PGE2 intrathecal injection significantly decreased GlyRα1 and GlyRα3 protein expressions in the L5 DH at 1 h and lasted to 5 h, and similar results were observed in the L5 DRG at 5 h. Confocal microscopic images showed the co-existence of punctate gephyrin and GlyRα3 immunoreactivity (IR) throughout the gray matter of the spinal cord, mainly in DH laminae I-III neurons and in ventral horn neurons. It also showed the co-existence of punctate gephyrin and GlyRα3 IR in DRG neurons. CONCLUSIONS: In this study, PGE2 intrathecal injection significantly decreased protein expression of gephyrin, GlyRα1 and GlyRα3 in spinal cord DH and DRG. The gephyrin and GlyRα3 were localized on neuron cells both in the DH and DRG.

Development of Antifouling Hyperbranched Polyglycerol Layers on Hydroxyl Poly-p-xylylene Coatings.

Antifouling surfaces that are resistant to protein adsorption and cell adhesion are desirable for many biomedical devices, such as diagnostic devices, biosensors, and implants. In this study, we developed an antifouling hyperbranched polyglycerol (hPG) surface on hydroxyl poly-p-xylylene (PPX-OH). PPX-OH was deposited via chemical vapor deposition (CVD), and an hPG film was then developed via the ring-opening reaction of glycidol. The hPG film greatly reduced the adhesion of L929 cells and platelets as well as protein adsorption. The addition of alkenyl groups in the hPG layer allows the conjugation of biomolecules, such as peptides and biotin, and elicits specific biological interactions. Since the CVD deposition of PPX-OH could be applied to most types of materials, our approach makes it possible to decorate an antifouling hPG film on most types of materials. Our method could be applied to biosensors, diagnostics, and biomedical devices in the future.

Posterior reversible encephalopathy as the first manifestation of Bickerstaff's brainstem encephalitis.

BACKGROUND: Posterior reversible encephalopathy syndrome (PRES) has been associated with Guillain-Barre syndrome in rare cases. Here we report a patient in whom PRES was the presenting manifestation of Bickerstaff's brainstem encephalitis. CASE PRESENTATION: A 75-year-old woman presented with acute onset of hypertension, headache, blurred vision, and left eyelid drooping. Magnetic resonance imaging of the brain showed characteristic PRES lesions involving the parietal and occipital lobes bilaterally. On the 6th day after symptom onset, the patient developed complete ptosis and external ophthalmoplegia of both eyes, progressive ataxia, and bilateral lower limb weakness. Cerebrospinal fluid analyses revealed albuminocytological dissociation (protein: 66.6 mg/dL, WBC: 0/µl), and nerve conduction studies showed demyelinating sensorimotor polyneuropathy. The patient developed somnolence and a left extensor plantar response on the 8th day. A diagnosis of Bickerstaff's brainstem encephalitis was made. Treatment with plasmapheresis led to a rapid improvement of clinical symptoms. To date, only five similar cases have been reported, but this is the only case in which PRES developed prior to treatment. CONCLUSIONS: PRES can be a comorbid condition with Bickerstaff's brainstem encephalitis, either preceding or following treatment; caution should be used in patients with either syndrome who exhibit atypical presentations.

Efficacy of continuous theta burst stimulation of the primary motor cortex in reducing migraine frequency: A preliminary open-label study.

BACKGROUND: Theta burst stimulation is a type of pattern-specific repetitive transcranial magnetic stimulation that requires less stimulation time and lower intensity to induce long-lasting effects comparable to those of other repetitive transcranial magnetic stimulation protocols. This pilot study investigated whether continuous theta burst stimulation (cTBS) on the primary motor cortex reduced headache frequency in patients with migraine. METHODS: Nine patients with migraine were recruited into our study. All patients received 20 cTBS sessions (bursts of 3 50-Hz TMS pulses at 200-ms intervals for 40 seconds), administered every weekday for 4 consecutive weeks. All patients kept headache diaries for 4 weeks before stimulation (baseline; T1), during stimulation (T2), and 4 weeks after stimulation (T3). The primary outcome measures were the changes of total headache and migraine days from baseline (Wilcoxon signed-rank test; T2 and T3 vs. T1). RESULTS: The number of total headache days was reduced at T2 and T3 compared with T1 [9.4 ± 6.2 days (p = 0.024) and 8.7 ± 10.1 days (p = 0.012) vs. 13.4 ± 10.1 days]. The number of migraine days was also reduced at T2 and T3 compared with T1 [2.9 ± 2.7 days (p = 0.021) and 1.0 ± 1.6 days (p = 0.008) vs. 8.6 ± 8.7 days]. CONCLUSION: Our results indicate that cTBS on the primary motor cortex might reduce the number of total headache and migraine days in patients with migraine. However, large-scale randomized controlled trials are necessary to further validate the findings.

Risk factors for levofloxacin resistance in Stenotrophomonas maltophilia from respiratory tract in a regional hospital.

OBJECTIVES: Stenotrophomonas maltophilia is a bacterial pathogen associated with health-care associated infections, particularly in immunocompromised patients. Members of the fluoroquinolone drug class are frequently used to treat S. maltophilia infection; however, S. maltophilia resistance to fluoroquinolones, especially levofloxacin, has been increasing. METHODS: We sought to identify risk factors associated with levofloxacin resistance using a case-control study. We examined sputum from 76 S. maltophilia-positive patients admitted to our hospital between January 1, 2010 and June 30, 2011. Case groups were defined as patients who had S. maltophilia infections resistant to levofloxacin, and control groups were defined as patients who had S. maltophilia infections susceptible to levofloxacin treatment. Patient information including demographics, previous antibiotic use, and other traits were recorded. In addition, S. maltophilia isolates from patient sputum were assessed for antibiotic resistance as well as for the presence of genes associated with drug resistance. RESULTS: Previous antibiotic treatment with first- or second-generation cephalosporin was found more often in the levofloxacin-susceptible group; by contrast, previous piperacillin/tazobactam treatment occurred more often in the levofloxacin-resistant group. Three genes associated with drug resistance, including SmeA, SmeD, and SpgM were not significantly different between these groups. CONCLUSION: Piperacillin/tazobactam treatment is associated with subsequent isolation of levofloxacin-resistant S. maltophilia from the respiratory tract.

Inhibition of peroxisome proliferator-activated receptor gamma prevents the melanogenesis in murine B16/F10 melanoma cells.

The purpose of this study was to investigate if PPARγ plays a role in the melanogenesis. B16/F10 cells were divided into five groups: control, melanin stimulating hormone (α-MSH), α-MSH+retinol, α-MSH+GW9662 (PPARγ antagonist), and GW9662. Cells in the control group were cultured in the Dulbecco's modified Eagle's medium (DMEM) for 48 hrs. To initiate the melanogenesis, cells in all α-MSH groups were cultured in medium containing α-MSH (10 nM) for 48 hrs. Cells were treated simultaneously with retinol (5 µM) in the α-MSH+retinol group. Instead of retinol, GW9662 (10 µM) was cocultured in the α-MSH+GW9662 group. Cells in the final group were cultured in the DMEM with GW9662. All the analyses were carried out 48 hours after treatments. The α-MSH was able to increase cell number, melanin production, and the activity of tyrosinase, the limiting enzyme in melanogenesis. These α-MSH-induced changes were prevented either by retinol or by GW9662. Further analyses of the activities of antioxidant enzymes including glutathione, catalase, and the superoxide dismutase (SOD) showed that α-MSH treatment raised the activity of SOD which was dependent on PPARγ level. According to our results, the α-MSH-induced melanogenesis was PPARγ dependent, which also modulated the expression of SOD.

Dose-dependent regulation of cell proliferation and collagen degradation by estradiol on ligamentum flavum.

BACKGROUND: Estradiol plays an important role in the regulation of collagen metabolism. Deficiency of estradiol has been reported to be associated with the degeneration of many connective tissues. However, the association of estradiol and hypertrophy of the ligamentum flavum was seldom explored. Therefore, we studied the effects of estradiol on cultured cells from the ligamentum flavum. METHODS: Primary cultures of human ligamentum flavum cells obtained from surgical specimens of 14 patients undergoing spinal surgery were used to investigate the effect of estradiol on cell proliferation and the expression of collagen, elastin, and matrix metalloproteinases. Downstream pathways of estrogen receptor underlying the regulation of metalloproteinases were also investigated. RESULTS: In our study, we revealed the existence of estrogen receptors on both female and male ligamentum flavum cells with a gender difference. 17ß-estradiol increased early (24 hours) proliferation of ligamentum flavum cells in a dose dependent manner and the effect could not be seen when the cell density increased. Estradiol with a concentration of 10(-9) M decreased collagen levels and increased the expression of MMP-13. Adding an antagonist of PI3K downstream pathway could reverse the expression of MMP-13 caused by estradiol. CONCLUSIONS: The results implied estradiol regulated the expression of MMP-13 via PI3K pathway and contributed to the homeostasis of extracellular matrix in the ligamentum flavum.

Requirement of inducible nitric-oxide synthase in lipopolysaccharide-mediated Src induction and macrophage migration.

Previously, we have demonstrated the induction of Src in lipopolysaccharide (LPS)-stimulated macrophages. In this study, we observed that pharmacological blockade or knockout of inducible nitric-oxide synthase (iNOS) reduced LPS-mediated Src induction and macrophage migration. Either SNAP (a NO donor) or 8-Br-cGMP (a cGMP analogue) could rescue these defects in iNOS-null macrophages, which indicated the participation of NO/cGMP in LPS-elicited Src expression and mobilization. In addition, Src family kinase (SFK)-specific inhibitor, PP2, inhibited SNAP- and 8-Br-cGMP-evoked motility implicating the involvement of SFKs downstream of NO/cGMP. Analysis of the expression of SFKs indicated LPS dramatically induced Src, which could be attributable to the increased level of the src transcript. Attenuation of Src by src-specific siRNA reduced LPS- and SNAP-evoked mobilization in Raw264.7 macrophages, and reintroduction of avian Src could rescue their motility. Furthermore, LPS-mediated Src induction led to increased FAK Pi-Tyr-397 and Pi-Tyr-861, which was also iNOS-dependent. With these findings, we concluded that iNOS was important for LPS-mediated macrophage locomotion and Src was a critical player in this process.

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase.

Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.

The role of N286 and D320 in the reaction mechanism of human dihydrolipoamide dehydrogenase (E3) center domain.

According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the K(cat) of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.

Gelatin-tricalcium phosphate membranes immobilized with NGF, BDNF, or IGF-1 for peripheral nerve repair: an in vitro and in vivo study.

In the present study, NGF, BNDF from the neurotrophin family and IGF-1 were covalently immobilized on gelatin-tricalcium phosphate (GTG) membrane using carbodiimide. We investigated the effects of these growth factors released from the GTG composites on cultured PC12 cells and sciatic nerve regeneration across a 10-mm-long gap in rats. In PC12 cell culture, the total protein content and MTT assay indicated more cell attachment on the composites modified with growth factors. The IGF-1 group showed a higher survival promotion effect on PC12 cells than did BDNF and NGF groups. On the other hand, NGF released from the composite showed the highest level of neuritogenesis for PC12 cells in neurite outgrowth assay. In the animal study, the GTG conduits modified with various growth factors were well tolerated by the host tissue. In the regenerated nerves, the number of the axons per unit area of the BDNF group was significantly higher than that of NGF and GTG groups but similar to that of IGF-1 group. However, the average axon size was the largest in NGF group. This result was in concordance with the neurite outgrowth assay in which NGF showed the highest neuritogenic potential. In the assessment of motor and sensory recovery after nerve repair, conduits modified with various neurotrophic factors showed a more favorable outcome in compound muscle action potential. The BDNF group had a better gastrocnemic muscle weight ratio than blank GTG repair. Nevertheless, the different effects of GTG conduits modified with various neurotrophic factors on functional recovery cannot be simply illustrated in the sciatic function index.

An in vivo study of tricalcium phosphate and glutaraldehyde crosslinking gelatin conduits in peripheral nerve repair.

In order to modulate the mechanical properties of gelatin, we previously developed a biodegradable composite composed by tricalcium phosphate and glutaraldehyde crosslinking gelatin (GTG) feasible for surgical manipulation. In this study, we evaluated the in vivo applications of GTG conduit for peripheral nerve repair. The effect of sciatic nerve reconstruction was compared between resorbable permeable GTG conduits and durable impermeable silicone tubes. Traditional methods of assessing nerve recovery following peripheral nerve repair including histomorphometric and electrophysiologic features were conducted in our study. In addition, autotomy score and sciatic function index (SFI) in walking tract analysis were used as additional parameters for assessing the return of nerve function. Twenty-four weeks after sciatic nerve repair, the GTG conduits were harvested. Microscopically, regeneration of nerves was observed in the cross-section at the mid portion of all implanted GTG conduits. The cross-sectional area of regenerated nerve of the GTG group was significant larger than that of the silicone group. In the compound muscle action potentials (CMAP), the mean recovery index of CMAP amplitude was 0.24 +/- 0.02 for the silicone group, 0.41 +/- 0.07 for the GTG group. The mean SFI increased with time in the GTG group during the evaluation period until 24 weeks. Walking tract analysis showed a higher SFI score in the GTG group at both 12 and 24 weeks. The difference reached a significant level at 24 weeks. Thus, the histomorphometric, electrophysiologic, and functional assessments demonstrate that GTG can be a candidate for peripheral nerve repair.

Release characteristics and bioactivity of gelatin-tricalcium phosphate membranes covalently immobilized with nerve growth factors.

The gelatin-tricalcium phosphate membranes were cross-linking with low concentration glutaraldehyde solution (GTG). This material has good mechanical property, biocompatibility, and is feasible for surgical manipulation. For axonal regeneration, nerve growth factors (NGF) were immobilized onto the composite (GTG) with carbodiimide. The purpose of this study was to evaluate the release characteristics and bioactivity of NGF after covalent immobilization onto the GTG membranes (GEN). NGF immobilized onto and released from the composite was quantified using ELISA method. PC 12 cells were cultured on the GTG and GEN composites. Cell survival, cytotoxicity, and cellular activity were evaluated by total protein content, LDH activity, and MTT assay respectively. Neurite outgrowth assay was used to evaluate the biological activity of NGF released from GEN composite. From ELISA measurement, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF were released from the composite in diffusion-controlled mechanism and degradation-controlled mechanism respectively. While culturing with PC 12 cells, LDH leakage results implied that whether GTG composite cross-linked with NGF or not showed little cytotoxicity. The total protein content and cellular activity of PC 12 cells were lower on GTG and GEN membranes than control group. However, 56%+/-3.98 of PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. In addition, sustained release of bioactive NGF for two months had been demonstrated by neurite outgrowth assay. From these experiments, it can be concluded that the technique used in the present study is capable of immobilizing NGF onto GTG membranes covalently and remaining the bioactivity of NGF. Therefore, GEN composite can be materials for sustained release of bioactive NGF and a candidate for future therapeutic application in nerve repair.

Novel CLCN1 mutations in Taiwanese patients with myotonia congenita.

We have performed genetic screening on the skeletal muscle chloride channel gene (CLCN1) in Taiwanese population. A total of four patients with myotonia congenita (MC) together with 106 normal individuals were examined. All 23 exons of the CLCN1 gene were analysed by direct sequencing of PCR products to detect the nucleotide changes. Five mutations and three polymorphisms were identified in this study. Among these, three missense mutations (S471F, P575S, D644G) and one polymorphism (T736I) are novel and could be unique to the Taiwanese. In addition, a previously documented recessive G482R mutation was identified in a heterozygous patient and his nonsymptomatic father, indicating that this mutation might indeed function recessively or dominantly with incomplete penetrance. In conclusion, this is the first report of MC in Taiwan with proven CLCN1 gene mutations and showing high molecular heterogeneity in Taiwanese MC patients.

Biocompatibility of NGF-grafted GTG membranes for peripheral nerve repair using cultured Schwann cells.

We previously developed a biodegradable composite with potentially good biocompatibility composed by tricalcium phosphate and gluataraldehyde cross-linking gelatin (GTG) with good mechanical property feasible for surgical manipulation. The purpose of this study was to evaluate the feasibility of immobilizing nerve growth factor (NGF) onto the composite (GTG) with carbodiimide (GEN composite). Cultured Schwann cells were seeded onto the GTG and GEN composites. For comparison, GTG membrane soaked in NGF solution without carbodiimide (GN composite) as cross-linking agent was also used to culture Schwann cells. Cell morphology was observed by a scanning electron microscope. Cell survival, cytotoxicity and cellular metabolism on the NGF-grafted GTG membrane were assessed quantitatively in terms of cell protein content, leakage of cytosolic lactate dehydrogenase (LDH) activity and by the well-established MTT assay, respectively. The result of LDH study did not show significant difference among GTG, NGF-modified GTG and control group. This indicated that GTG composite, whether cross-linking with NGF or not, has little cytotoxic effect. Comparing the protein content and MTT assay among GEN, GN composite and control group, the data confirmed more attachment of Schwann cells on GEN composite. Although GTG cross-linking with NGF did not promote Schwann cell proliferation, the techniques we used in this study provided a method to fabricate a novel biomaterial incorporation of Schwann cells and covalently immobilized NGF.