Radionuclide Reduction by Combinatorial Optimization of Microbial Extracellular Electron Transfer with a Physiologically Adapted Regulatory Platform.

Microbial extracellular electron transfer (EET) is the basis for many microbial processes involved in element geochemical recycling, bioenergy harvesting, and bioremediation, including the technique for remediating U(VI)-contaminated environments. However, the low EET rate hinders its full potential from being fulfilled. The main challenge for engineering microbial EET is the difficulty in optimizing cell resource allocation for EET investment and basic metabolism and the optimal coordination of the different EET pathways. Here, we report a novel combinatorial optimization strategy with a physiologically adapted regulatory platform. Through exploring the physiologically adapted regulatory elements, a 271.97-fold strength range, autonomous, and dynamic regulatory platform was established for Shewanella oneidensis, a prominent electrochemically active bacterium. Both direct and mediated EET pathways are modularly reconfigured and tuned at various intensities with the regulatory platform, which were further assembled combinatorically. The optimal combinations exhibit up to 16.12-, 4.51-, and 8.40-fold improvements over the control in the maximum current density (1009.2 mA/m2) of microbial electrolysis cells and the voltage output (413.8 mV) and power density (229.1 mW/m2) of microbial fuel cells. In addition, the optimal strains exhibited up to 6.53-fold improvement in the radionuclide U(VI) removal efficiency. This work provides an effective and feasible approach to boost microbial EET performance for environmental applications.

Epigallocatechin Gallate Attenuates Gentamicin-Induced Nephrotoxicity by Suppressing Apoptosis and Ferroptosis.

Gentamicin (GEN) is a kind of aminoglycoside antibiotic with the adverse effect of nephrotoxicity. Currently, no effective measures against the nephrotoxicity have been approved. In the present study, epigallocatechin gallate (EG), a useful ingredient in green tea, was used to attenuate its nephrotoxicity. EG was shown to largely attenuate the renal damage and the increase of malondialdehyde (MDA) and the decrease of glutathione (GSH) in GEN-injected rats. In NRK-52E cells, GEN increased the cellular ROS in the early treatment phase and ROS remained continuously high from 1.5 H to 24 H. Moreover, EG alleviated the increase of ROS and MDA and the decrease of GSH caused by GEN. Furthermore, EG activated the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). After the treatment of GEN, the protein level of cleaved-caspase-3, the flow cytometry analysis and the JC-1 staining, the protein levels of glutathione peroxidase 4 (GPX4) and SLC7A11, were greatly changed, indicating the occurrence of both apoptosis and ferroptosis, whereas EG can reduce these changes. However, when Nrf2 was knocked down by siRNA, the above protective effects of EG were weakened. In summary, EG attenuated GEN-induced nephrotoxicity by suppressing apoptosis and ferroptosis.

Enhanced Bioreduction of Radionuclides by Driving Microbial Extracellular Electron Pumping with an Engineered CRISPR Platform.

Dissimilatory metal-reducing bacteria (DMRB) with extracellular electron transfer (EET) capability show great potential in bioremediating the subsurface environments contaminated by uranium through bioreduction and precipitation of hexavalent uranium [U(VI)]. However, the low EET efficiency of DMRB remains a bottleneck for their applications. Herein, we develop an engineered CRISPR platform to drive the extracellular electron pumping of Shewanella oneidensis, a representative DMRB species widely present in aquatic environments. The CRISPR platform allows for highly efficient and multiplex genome editing and rapid platform elimination post-editing in S. oneidensis. Enabled by such a platform, a genomic promoter engineering strategy (GPS) for genome-widely engineering the EET-encoding gene network was established. The production of electron conductive Mtr complex, synthesis of electron shuttle flavin, and generation of NADH as intracellular electron carrier are globally optimized and promoted, leading to a significantly enhanced EET ability. Applied to U(VI) bioreduction, the edited strains achieve up to 3.62-fold higher reduction capacity over the control. Our work endows DMRB with an enhanced ability to remediate the radionuclides-contaminated environments and provides a gene editing approach to handle the growing environmental challenges of radionuclide contaminations.

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria.

The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real-world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the intron-encoded homing-endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR-1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome-editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow-up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.

Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis.

The unique extracellular electron transfer (EET) ability has positioned electroactive bacteria (EAB) as a major class of cellular chassis for genetic engineering aimed at favorable environmental, energy, and geoscience applications. However, previous efforts to genetically enhance EET ability have often impaired the basal metabolism and cellular growth due to the competition for the limited cellular resource. Here, we design a quorum sensing-based population-state decision (PSD) system for intelligently reprogramming the EET regulation system, which allows the rebalanced allocation of the cellular resource upon the bacterial growth state. We demonstrate that the electron output from Shewanella oneidensis MR-1 could be greatly enhanced by the PSD system via shifting the dominant metabolic flux from initial bacterial growth to subsequent EET enhancement (i.e., after reaching a certain population-state threshold). The strain engineered with this system achieved up to 4.8-fold EET enhancement and exhibited a substantially improved pollutant reduction ability, increasing the reduction efficiencies of methyl orange and hexavalent chromium by 18.8- and 5.5-fold, respectively. Moreover, the PSD system outcompeted the constant expression system in managing EET enhancement, resulting in considerably enhanced electron output and pollutant bioreduction capability. The PSD system provides a powerful tool for intelligently managing extracellular electron transfer and may inspire the development of new-generation smart bioelectrical devices for various applications.

Rediverting Electron Flux with an Engineered CRISPR-ddAsCpf1 System to Enhance the Pollutant Degradation Capacity of Shewanella oneidensis.

Pursuing efficient approaches to promote the extracellular electron transfer (EET) of extracellular respiratory bacteria is essential to their application in environmental remediation and waste treatment. Here, we report a new strategy of tuning electron flux by clustered regularly interspaced short palindromic repeat (CRISPR)-ddAsCpf1-based rediverting (namely STAR) to enhance the EET capacity of Shewanella oneidensis MR-1, a model extracellular respiratory bacterium widely present in the environment. The developed CRISPR-ddAsCpf1 system enabled approximately 100% gene repression with the green fluorescent protein (GFP) as a reporter. Using a WO3 probe, 10 representative genes encoding for putative competitive electron transfer proteins were screened, among which 7 genes were identified as valid targets for EET enhancement. Repressing the valid genes not only increased the transcription level of the l-lactate metabolism genes but also affected the genes involved in direct and indirect EET. Increased riboflavin production was also observed. The feasibility of this strategy to enhance the bioreduction of methyl orange, an organic pollutant, and chromium, a typical heavy metal, was demonstrated. This work implies a great potential of the STAR strategy with the CIRPSR-ddAsCpf1 system for enhancing bacterial EET to favor more efficient environmental remediation applications.

Early initiation of argatroban therapy in the management of acute superior mesenteric venous thrombosis.

Acute superior mesenteric venous thrombosis (ASMVT) is an intractable disease with poor prognosis. Argatroban, a direct thrombin inhibitor, may be a novel anticoagulant method in the therapy of ASMVT. The aim of the present study was to assess the efficacy and safety of early argatroban therapy in ASMVT patients. The current retrospective study reviewed a consecutive series of ASMVT patients receiving early argatroban therapy during hospitalization between March 2013 and April 2014, with 18 ASMVT patients included in the study. Of these, 16 patients without hepatic dysfunction underwent anticoagulant therapy with argatroban with a mean dose of 1.57±0.34 µg/kg/min and a mean duration of 12.2±3.7 days, while their activated partial thromboplastin time (aPTT) was elevated to 1.95±0.26 times the baseline value. In addition, 2 hepatic dysfunction patients received therapy with a dose of 0.41 µg/kg/min and 0.46 µg/kg/min, and with aPTT of 1.68 and 1.62 times the baseline value, respectively. Overall, 94% (n=17) of the patients presented clinical improvement, while 88% (n=16) of patients presented partially or completely dissolved thrombus in contrast-enhanced computed tomography images. The incidence of surgery and bowel resection was 6% (excluding 1 case with intestinal necrosis detected on admission). Furthermore, 11% (n=2) of patients experienced a bleeding episode, however no major bleeding or mortality occurred during hospitalization. During the follow-up, the mortality and the recurrence rate were 6% and 11%, respectively. In conclusion, early initiation of argatroban treatment may be an effective and safe therapy in ASMVT, manifesting efficient resolution of the thrombus, rapid improvement of symptoms, low incidence of bowel resection and bleeding complication, and low mortality rate.

[The role of SHH pathway in PDGF-induced VSMC proliferation].

OBJECTIVE: To discuss the role of sonic hedgehog (SHH) pathway in PDGF-induced vascular smooth muscle cell proliferation. METHOD: Human vein VSMC were cultured in vitro. Laser confocal microscopy and Western blot were used to detect the expression of SHH pathway related proteins. The cell proliferation was evaluated by Ki-67 staining and BrdU incorporation after treatment by siRNA of Gli2 or shh pathway inhibitor cyclopamine. RESULTS: The results of laser confocal microscopy and Western blot showed that SHH pathway protein which including shh, Patched1 and Gli2 were activated in the PDGF-induced VSMC proliferation.BrdU incorporation assay and Ki-67 staining showed that the cell proliferation which induced by PDGF was inhibited by Gli2-siRNA and cyclopamine which can both block SHH pathway. CONCLUSION: SHH pathway play an important role in PDGF-induced VSMC proliferation.

[Evaluation of exposure and sensitization to house dust mites among rhinitis patients in the steppe environment of Inner Mongolia].

OBJECTIVE: To evaluate the correlation of exposure and sensitization to house dust mites among rhinitis patients, and the consistency of skin prick test (SPT) and serum specific IgE (sIgE) in the detection of sensitization in a steppe environment of inner Mongolia. METHODS: Three hundreds and fourteen patients with rhinitis symptoms were recruited in this study in a hospital setting at Xilinhot City, inner Mongolia. All patients underwent medical history investigation, anterior rhinoscopy, and allergen tests in vivo (SPT) and in vitro (sIgE). Dust samples were collected from mattresses in patient's bedroom. The concentrations of group I major allergen of Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) were measured by enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed using a SPSS 17.0 software (t test, χ(2) test and so on). RESULTS: Among a total of 314 patients, the prevalence of positive SPT for Der p and Der f was 5.7% and 22.0%, respectively; and the prevalence of positive sIgE for Der p and Der f was 9.2% and 7.6%, respectively. Sensitization rates for house dust mites (Der p and/or Der f) were lower than those of pollens. The pollen allergen with the highest positive rate was mugwort (SPT, 51.9%; sIgE, 47.1%). Diagnostic tests using SPT as the gold standard for sensitization showed that the positive likelihood ratio of sIgE was 4.27 for Der p and 10.64 for Der f, and the Kappa value was 0.20 for Der p and 0.35 for Der f, respectively. A total of 276 dust samples collected from patient's mattresses were measured. The concentrations of Der p 1 and Der f1 were detectable in 2 (0.7%) and 4 (1.4%) mattress samples, respectively. There was positive correlation between the exposure levels of Derp 1 and the SPT positive reaction to Der p (r(s) = 0.156, P = 0.01), but no significant correlation was found between the exposure levels of Der p 1 and the sIgE positive reaction to Der p (r(s) = 0.116, P = 0.055). There was no significant correlation between the exposure levels of Der f 1 and the SPT as well as sIgE positive reaction to Der f 1(r(s) = 0.05, P = 0.931; r(s) = 0.07, P = 0.245). CONCLUSIONS: In the steppe environment of inner Mongolia, exposure to house dust mites are very low, and the Der p and Der f are not the major allergens in patients with allergic rhinitis. Also, it was not a simple dose-response relationship between exposure and sensitization to house dust mites. In such a specific environment, there is lower consistency between the results of SPT and sIgE in the detection of sensitization to house dust mites. Therefore, it is recommended that the allergen testing in vivo and in vitro should be combined for clinical diagnosis.

[The study about expression of Gli2 in vein grafts].

OBJECTIVE: To study the expression of cell cycle related factor Gli2 in autogenous vein graft and its relation with neointima formation. METHOD: Autogenous vein graft model were established in 36 male wistar rats of 8 weeks old, 140 g, by transplanting the left jugular vein to intra renal abdominal aorta with microsurgical technique. Graft veins were harvest at 14, 28 days after transplantation. The IF and W-B were used to detect the protein expression in the vein graft. At the same time Gli2- mRNA was measured by RT-PCR. RESULTS: Immunofluorescent staining showed that the Gli2+ cells was only 3.2% ± 0.4% in the normal vein, but was much more in the vein graft after surgery, was 41.3% ± 0.6%, 58.3 ± 0.6% respectively; The expression of Gli2 and PCNA were both elevated in the vein graft. There is a positive correlation between them which indicated by W-B, the relation index was 0.826; the Gli2 mRNA content was also increased in vein graft, was 8.9, 13.6 fold compared with normal vein as 1 respectively. CONCLUSION: Gli2 is upregulated in autogenous vein grafts and may correlated with the proliferation of vascular smooth muscle cells.

[The expression of sonic hedgehog in rat vein grafts].

OBJECTIVE: To study the expression of cell cycle related factor sonic hedgehog (SHH) in autogenous vein graft and its relation with neointima formation. METHODS: Autogenous vein graft model were established in 24 male Wistar rats of 8 weeks old and 140 g weight, by transplanting the left jugular vein to intra renal abdominal aorta with microsurgical technique. Graft veins were harvested at 14 d and 28 d after transplantation. The immunohistochemistry and Western blot were used to detect the SHH and PCNA expression in the vein graft. At the same time SHH mRNA was measured by quantitative real-time PCR. The opposite normal veins served as control. RESULTS: Histological staining showed that the percent of SHH+ cells was only (2.0 +/- 0.5)% in the normal vein, but was much more in the vein graft after surgery, as (39.4 +/- 0.4)% and (63.0 +/- 0.3)% respectively (P < 0.01). The expression of SHH and PCNA were both elevated in the vein graft. There was a positive correlation between them which indicated by Western blot (r = 0.808, P < 0.01). The SHH mRNA content also increased in vein graft to 9.5 and 23.8 folds of that in control. CONCLUSION: SHH is upregulated in autogenous vein grafts and may correlated with the proliferation of vascular smooth muscle cells.

[The sonic hedgehog induce vascular adventitial fibroblasts phenotypic modulation, proliferation and migration].

OBJECTIVE: To study the effect of shh on the migration, proliferation and phenotypic modulation of vascular adventitial fibroblasts. METHOD: Cultivate the vascular adventitial fibroblast in vitro. Use immunofluorescent, laser confocal microscopy, Western-blot and real-time PCR to detect the expression of mRNA and protein of related index. Estimate cell proliferation according to the expression of Ki67 and cell proliferation curve. Application of wound healing test to estimate migration of fibroblast. The expression of alpha-actin is thought to be marker of phenotypic modulation of fibroblast. RESULT: The expression of shh was detected in vascular adventitial fibroblast in vitro. After addition of exogenous shh (3.5 microg/ml), there were more Ki67(+) cells and the wounding area which was covered by cells became larger. The expression of alpha-actin was detected. After addition of cyclopamine (40 micromol/L), there were less Ki67(+) cells and the wounding area which was covered by cells became smaller. CONCLUSION: Shh can promote proliferation, migration and phenotypic modulation of vascular adventitial fibroblasts.