Optogenetic Activation of Peripheral Somatosensory Neurons in Transgenic Mice as a Neuropathic Pain Model for Assessing the Therapeutic Efficacy of Analgesics.

Optogenetics is a novel biotechnology widely used to precisely manipulate a specific peripheral sensory neuron or neural circuit. However, the use of optogenetics to assess the therapeutic efficacy of analgesics is elusive. In this study, we generated a transgenic mouse stain in which all primary somatosensory neurons can be optogenetically activated to mimic neuronal hyperactivation in the neuropathic pain state for the assessment of analgesic effects of drugs. A transgenic mouse was generated using the advillin-Cre line mated with the Ai32 strain, in which channelrhodopsin-2 fused to enhanced yellow fluorescence protein (ChR2-EYFP) was conditionally expressed in all types of primary somatosensory neurons (advillincre/ChR2+/+). Immunofluorescence and transdermal photostimulation on the hindpaws were used to verify the transgenic mice. Optical stimulation to evoke pain-like paw withdrawal latency was used to assess the analgesic effects of a series of drugs. Injury- and pain-related molecular biomarkers were investigated with immunohistofluorescence. We found that the expression of ChR2-EYFP was observed in many primary afferents of paw skin and sciatic nerves and in primary sensory neurons and laminae I and II of the spinal dorsal horns in advillincre/ChR2+/+ mice. Transdermal blue light stimulation of the transgenic mouse hindpaw evoked nocifensive paw withdrawal behavior. Treatment with gabapentin, some channel blockers, and local anesthetics, but not opioids or COX-1/2 inhibitors, prolonged the paw withdrawal latency in the transgenic mice. The analgesic effect of gabapentin was also verified by the decreased expression of injury- and pain-related molecular biomarkers. These optogenetic mice provide a promising model for assessing the therapeutic efficacy of analgesics in neuropathic pain.

Characteristics of laser-induced steel plasmas generated with different focusing conditions.

Laser focusing is an important parameter that affects the characteristics of laser-induced plasma. Focusing lenses with different F-numbers form different energy density distributions near the surface of a sample, thus affecting the characteristics of plasma. In this study, the plasma generated by a nanosecond laser ablation of a micro-alloy steel certified sample at 1 atm of air was investigated. We compare the spectrally integrated plasma images obtained at different defocusing distances for short- and long-focus lenses and investigate the optical emission spectra of laser-induced plasma on steel alloy by using focusing lenses with different F-numbers. With an increase in the defocusing distance, the plasma plume changes from flat to hemispherical and then splitting occurs. The spectral line intensity increases first and then decreases, then increases slightly, and finally decreases gradually. For the long-focus lens, when the focal point is above the sample surface, the laser beam strongly interacts with air over a longer distance, leading to longer air plasma and weaker sample plasma compared with the short-focus lens. Thus, the relative intensity of the second peak in the spectral line intensity, according to the defocusing distance, gradually decreases with increasing F-number. We also obtain two-dimensional spatial distributions of the spectral line intensity according to the F-number and defocusing distance. The optimal defocusing distances for all focusing lenses occur when the focal point is below the sample surface. The relation between the optimal defocusing distance and F-number follows a single- exponential decay function.

Weak Metal-Support Interaction over CuO/TiO2 Catalyst Governed Low-Temperature Toluene Oxidation.

Regulating the metal-support interaction is essential for obtaining highly efficient catalysts for the catalytic oxidation of volatile organic compounds (VOCs). In this work, CuO-TiO2(coll) and CuO/TiO2(imp) with different metal-support interactions were prepared via colloidal and impregnation methods, respectively. The results demonstrated that CuO/TiO2(imp) has higher low-temperature catalytic activity, with a 50% removal of toluene at 170 °C compared to CuO-TiO2(coll). Additionally, the normalized reaction rate (6.4 × 10-6 mol·g-1·s-1) at 160 °C over CuO/TiO2(imp) was almost four-fold higher than that over CuO-TiO2(coll) (1.5 × 10-6 mol·g-1·s-1), and the apparent activation energy value (27.9 ± 2.9 kJ·mol-1) was lower. Systematic structure and surface analysis results disclosed that abundant Cu2+ active species and numerous small CuO particles were presented over CuO/TiO2(imp). Owing to the weak interaction of CuO and TiO2 in this optimized catalyst, the concentration of reducible oxygen species associated with the superior redox property could be enhanced, thus significantly contributing to its low-temperature catalytic activity for toluene oxidation. This work is helpful in exploring the influence of metal-support interaction on the catalytic oxidation of VOCs and developing low-temperature catalysts for VOCs catalytic oxidation.

Corrigendum: Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China.

[This corrects the article DOI: 10.3389/fmicb.2022.1018682.].

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China.

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr-1 (1.10%, 2/181), tet(X4) (1.10%, 2/181), and cfr (6.08%, 2/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified astA (37.02%, 67/181), hlyA (1.66%, 3/181), hlyB (1.66%, 3/181) and hlyD (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Effect of ß-1,3/1,6-glucan on gut microbiota of yellow-feathered broilers.

ß-1,3/1,6-glucan as a prebiotic improves immune performance in animals. These functions are closely related to the effect of ß-1,3/1,6-glucan on gut microbiota structure. However, the effect of ß-1,3/1,6-glucan on the gut microbiota structure of broilers is unclear. The aim of this study was to confirm the effects of ß-1,3/1,6-glucan on the cecal microflora structure of yellow-feathered broilers. This study monitored the antimicrobial resistance (AMR) level of Escherichia coli in feces of yellow-feathered broilers by standard broth dilution method and mastered the AMR level of chickens selected. The effects of ß-1,3/1,6-glucan on gut microbiota were investigated by 16S rRNA sequencing. The results showed that the number of isolated multidrug-resistant E. coli strains accounted for 98.41%. At 14, 21, and 28 days of age, supplemented of 0.2%, 0.1%, and 0.1% ß-1,3/1,6-glucan in yellow-feathered broiler diets significantly altered gut microbial composition, and beneficial bacteria Alistipes, Bacteroides and Faecalibacterium were significantly increased. These findings provide guidance and recommendations for ß-1,3/1,6-glucan as a broiler feed additive to improve the growth of broilers.

Antimicrobial resistance surveillance of Escherichia coli from chickens in the Qinghai Plateau of China.

Antimicrobial resistance (AMR) may lead to worldwide epidemics through human activities and natural transmission, posing a global public safety threat. Colistin resistance mediated by the mcr-1 gene is the most prevalent among animal-derived Escherichia coli, and mcr-1-carrying E. coli have been frequently detected in central-eastern China. However, animal-derived E. coli with AMR and the prevalence of mcr-1 in the Qinghai Plateau have been rarely investigated. Herein, 375 stool samples were collected from 13 poultry farms in Qinghai Province and 346 E. coli strains were isolated, of which eight carried mcr-1. The AMR rates of the E. coli strains to ampicillin, amoxicillin/clavulanic acid, and tetracycline were all above 90%, and the resistance rates to ciprofloxacin, cefotaxime, ceftiofur, and florfenicol were above 70%. Multidrug-resistant strains accounted for 95.66% of the total isolates. Twelve E. coli strains showed colistin resistance, from which a total of 46 AMR genes and 36 virulence factors were identified through whole-genome sequencing. The mcr-1 gene resided on the IncHI2, IncI2-type and IncY-type plasmids, and mcr-1 was located in the nikA-nikB-mcr-1-pap2 gene cassette (three strains) or the pap2-mcr-1-ISApl1 structure (one strain). Completed IncI2-type plasmid pMCR4D31-3 sequence (62,259 bp) revealed that it may cause the horizontal transmission of mcr-1 and may increase the risk of its spread through the food chain. Taken together, the AMR of chicken-derived E. coli in the plateau is of concern, suggesting that it is very necessary for us to strengthen the surveillance in various regions under the background of one health.

The effect of bile salt diet supplementation on genes related to fat metabolism in yellow-feathered broilers.

Background and Aim: As a new feed additive, bile acid (BA) can promote the absorption and transport of lipids and fat-soluble vitamins. In recent years, BAs have been widely used in animal feed to promote fat absorption. Therefore, this study aimed to investigate the effect of bile salt supplementation in the diet of yellow-feathered broilers on messenger RNA (mRNA) expression of sterol regulatory element-binding protein 1 (SREBF1), fatty acid synthase (FAS), acetyl-coenzyme A carboxylase (ACC), and fatty acid transport protein 4 (FATP4). Materials and Methods: Four hundred and twenty commercial male chicks were randomly divided into seven groups (with four replicates per group and 15 chickens per replicate). They were fed diets supplemented with bile salts at 0, 1.5, 2.5, 3.5, 4.5, 5.5 mg/kg, and 2 mg/kg tylosin for 30 days. Changes in SREBF1, fatty acid transporter 4, FAS, and acetyl-CoA carboxylase genes in intestinal mucosa and liver of yellow-feathered broilers were determined using a quantitative fluorescence polymerase chain reaction. Results: mRNA expression of SREBF1, FAS, ACC, and FATP4 in the small intestine decreased in chicks fed diets supplemented with 3.5 and 4.5 mg/kg bile salts (p<0.05) compared with the control group on 7 days and 14 d. The mRNA expressions of SREBF1, FAS, ACC, and FATP4 in liver tissue decreased in chicks fed diets supplemented with 4.5 and 5.5 mg/kg bile salts (p<0.05) compared to the control group on 7 days. The mRNA expression of SREBF1, FAS, ACC, and FATP4 in the liver at 14 days and the small intestine on 21 days also decreased in chicks fed diets supplemented with 4.5 mg/kg bile salts (p<0.05) compared to the control group. When contrasted with the control group on day 21, the mRNA expression of SRWBF1, FAS, ACC, and FATP4 detected in the liver was lower in chicks fed diets supplemented with bile salts (p<0.05). Conclusion: The dietary supplementation of bile salts at 4.5 mg/kg effectively regulates the expression of fat metabolism genes, such as SREBF1, FAS, ACC, and FATP4 mRNA. At this concentration, bile salts promote fat catabolism, inhibit fat synthesis, and play an essential role in improving the fat deposition of broilers.

The effectiveness of travel restriction measures in alleviating the COVID-19 epidemic: evidence from Shenzhen, China.

With the expansion of the global novel coronavirus disease (COVID-19) pandemic, unprecedented interventions have been widely implemented in many countries, including China. In view of this scenario, this research aims to explore the effectiveness of population mobility restriction in alleviating epidemic transmission during different stages of the outbreak. Taking Shenzhen, a city with a large immigrant population in China, as a case study, the real-time reproduction number of COVID-19 is estimated by statistical methods to represent the dynamic spatiotemporal transmission pattern of COVID-19. Furthermore, migration data between Shenzhen and other provinces are collected to investigate the impact of nationwide population flow on near-real-time dynamic reproductive numbers. The results show that traffic flow control between populated cities has an inhibitory effect on urban transmission, but this effect is not significant in the late stage of the epidemic spread in China. This finding implies that the government should limit international and domestic population movement starting from the very early stage of the outbreak. This work confirms the effectiveness of travel restriction measures in the face of COVID-19 in China and provides new insight for densely populated cities in imposing intervention measures at various stages of the transmission cycle.

Spin-orbit Hall effect in the tight focusing of a radially polarized vortex beam.

When the first-order radially polarized vortex beam propagates in an uniaxial crystal, the spin and the orbital angular momentum parts can be separated. It is called the optical spin-orbit Hall effect. In this study, we investigate the tight focusing of the radially polarized vortex beam theoretically and find the spatial separation of the spin and the orbital angular momentum parts occurs in the focal plane when the polarization order equals 1 and the vortex charge equals 1 (or -1). Moreover, when the initial phase of the polarization state takes π/2, the spatial separation of intensity in the focal plane corresponds to the spatial separation of the spin and the orbital angular momentum parts. This phenomenon can be considered as a manifestation of the optical spin-orbit Hall effect in the tight focusing of radially polarized vortex beam. Also, we show that, when the polarization order is greater than 1, the initial phase change of polarization state just leads to the rotation of the focal field and the spin and the orbital angular momentum density in the focal plane. Our results provide the potential application in the field of optical micro-manipulation.

The effect of Aspergillus niger as a dietary supplement on blood parameters, intestinal morphology, and gut microflora in Haidong chicks reared in a high altitude environment.

AIM: The effects of the inclusion of Aspergillus niger in the diet of Haidong chicks reared in the Qing-Zang high altitude area (China) under hypoxic conditions. MATERIALS AND METHODS: A total of 720 Haidong chicks were randomly divided into six groups and fed diets supplemented with 0%, 0.5%, 0.75%, 1.0%, 1.25%, and 1.5% of A. niger to determine blood parameters, intestinal morphology, and gut microflora in Haidong chicks reared in a high altitude environment. RESULTS: Packed cell volume (PCV), red blood cell (RBC) count, white blood cell count, and hemoglobin concentration increased in the groups fed diets containing A. niger. The administration of A. niger in 1.0% and 1.25% significantly decreased the concentration of Escherichia coli in the cecum, while the concentration of Bifidobacterium and Lactobacillus in the cecum and ileum was increased in the treated groups. When compared to the control groups, villi height, crypt depth, and goblet cell density in the intestine was raised, in general, in the groups treated with A. niger. CONCLUSION: These findings suggest that 1.25% A. niger as dietary supplement may improve the resistance to ascites among birds reared under hypoxic conditions.

Excessive Se on RuSe2 nanocrystals to accelerate water dissociation for the enhanced electrocatalytic hydrogen evolution reaction.

Selenium-enriched RuSe2 (RuxSe) nanocrystals as electrocatalysts for the HER in basic media have been synthesized via a facile hydrothermal method followed by a calcination process. The catalytic activity of the obtained RuxSe nanocrystals is greatly dependent on calcination temperatures. The nanocrystals obtained at 400 °C (RuxSe-400) demonstrate the highest HER activity with a low overpotential of 45 mV to deliver a current density of 10 mA cm-2 and a small Tafel slope of 31.4 mV dec-1. The enhanced catalytic HER performance of RuxSe-400 could be attributed to the excessive Se on the RuSe2 nanocrystal surface. Density functional theory (DFT) calculations reveal that the excessive Se would lower the energy barrier for water dissociation and lessen the dependence on the Ru sites for OH* adsorption but have a negligible effect on hydrogen adsorption energy, leading to an accelerated HER process. Furthermore, the excessive Se on the nanocrystal surface further endows the catalyst with promoted charge-transfer kinetics, ensuring a more efficient catalytic reaction. The strategy herein for the design of highly efficient HER catalysts by engineering the separation of different intermediate (H* and OH*) adsorption sites is expected to be extended to other electrocatalysts for high-efficiency energy conversion.

[Low-magnitude vibration promotes osteogenesis of osteoblasts in ovariectomized osteoporotic rats via the estrogen receptor α].

The purpose of this study was to investigate the effect of low-magnitude vibration on osteogenesis of osteoblasts in ovariectomized rats with osteoporosis via estrogen receptor α(ERα). The mRNA expression of osteogenic markers were examined with qRT-PCR, based on which the optimal vibration parameter for promoting osteogenesis was determined (45 Hz × 0.9 g, g = 9.8 m/s2). Then we loaded the optimal vibration parameter on the osteoblasts of ovariectomized rats with osteoporosis. The protein expression of osteogenic markers and ERα were detected with Western blot; the distribution of ERα was examined with immunofluorescence technique. Finally, through inhibiting the expression of ERα with estrogen receptor inhibitor ICI182780, the protein and mRNA expression of osteogenic markers were examined. First, the results showed that low-magnitude vibration could promote the expression of osteogenic markers and ERα in osteoblasts of ovariectomized rats with osteoporosis (P < 0.05), and make ERα transfer to the nucleus. On the other hand, the results also showed that after inhibiting the expression of ERα in osteoblasts of ovariectomized rats with osteoporosis, the protein and mRNA expression of osteogenic marker were decreased (P < 0.05). In our study, low-magnitude vibration played an important role in the osteogenesis of osteoblasts in ovariectomized rats with osteoporosis through increasing the expression and causing translocation of ERα. Furthermore, it provides a theoretical basis for the application of low-magnitude vibration in the prevention and treatment of postmenopausal osteoporosis.

Converting Escherichia coli MG1655 into a chemical overproducer through inactivating defense system against exogenous DNA.

Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production. However, the direct application of this strain suffers from the transformation inefficiency and plasmid instability. Herein, we conducted genetic modifications at a serial of loci of MG1655 genome, generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways. Using JW128 as the host, the titer of isobutanol reached 5.76 g/L in shake-flask fermentation, and the titer of lycopene reached 1.91 g/L in test-tube fermentation, 40-fold and 5-fold higher than that of original MG1655, respectively. These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering.

Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering. KEY POINTS: • This article reports an efficient genome engineering tool for E. coli. • The tool is advantageous for the manipulations of long DNA fragments. • The tool has been successfully applied for genome simplification. • The tool has been successfully applied for metabolic engineering.

[Screening of the Optimum Medium for Rat Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of three different cell culture mediums, DMEM-LG, α-MEM and DMEM/F12, on the growth of rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and so that to screen out the most suitable medium for in vitro culturing the rat BMSCs. METHODS: BMSCS were isolated from the femur and tibia of SD rats by whole bone marrow differential adherence method. The isolated cells were then cultured with three culture mediums, DMEM-LG, α-MEM and DMEM/F12. The rat BMSCs morphology, adhesion, proliferation, the time of passage and the number the colony at day 14 in three mediums respectively were observed with inverted phase contrast microscopy and compared. Flow cytometry was used to identify and observe the effects of different mediums on the surface antigen expression of rats BMSCs. RESULTS: Compared with the other two groups of media, BMSCs cultured in DMEM-LG had shorter colony formation time, shorter first passage time, more clone formation (14±2) and showed uniform morphology and the highest attachment efficiency (47.0±2.8)%. Meanwhile, BMSCs cultured with DMEM-LG entered logarithmic growth phase after only 4 days of culturing and showed the highest average specific growth rate and the largest average number of propagations per unit time. The total number of cells reached about (2.2-2.7)×105 mL-1 within three days. The cells cultured with 3 mediums were all identified as rat BMSCs, and the expression of surface antigen in BMSCs was not significantly affected by different media. CONCLUSION: DMEM-LG is more suitable for proliferation of rat BMSCs in vitro.

CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli.

Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. coli) rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in genetic engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

Optical vortex shaping via a phase jump factor.

Topological charge (TC) of an optical vortex (OV) is a crucial parameter. We propose two factors, namely, the phase jump factor and the phase gradient factor, to replace the parameter of TC through unwrapping the TC definition integral. Based on these two factors, we report on a novel OV, referred to as the remainder-phase optical vortex (ROV). The properties of the ROV are studied in depth by adjusting these two factors. Results show that the phase gradient factor determines the total orbital angular momentum (OAM), whereas the phase jump factor decides the number of split unit vortices and reshapes the structure of the OAM distribution. This work provides a novel OV with controllable OAM distribution, which will open up new applications such as particle manipulation, beam shaping, and micro-fabrication.

Ru x Se@MoS2 hybrid as a highly efficient electrocatalyst toward hydrogen evolution reaction.

Alkaline hydrogen evolution reaction (HER) requires highly efficient and stable catalytic materials, the engineering of which needs overall consideration of the water dissociation process as well as the intermediate hydrogen adsorption process. Herein, a Ru x Se@MoS2 hybrid catalyst was synthesized by the decoration of MoS2 with Ru x Se nanoparticles through a two-step hydrothermal reaction. Due to the bifunctionality mechanism in which Ru promotes the water dissociation and the nearby Se atoms, unsaturated Mo and/or S atoms act as active sites for the intermediate hydrogen adsorption, the hybrid catalyst exhibits an exceptional HER performance in basic media with a rather low overpotential of 45 mV at a current density of 10 mA cm-2 and a small Tafel slope of 42.9 mV dec-1. The synergetic effect between Ru x Se and MoS2 not only enables more catalytically active sites, but also increases the inherent conductivity of the hybrid catalyst, leading to more favorable HER kinetics under both alkaline and acidic conditions. As a result, Ru x Se@MoS2 also demonstrates an enhanced catalytic activity toward HER in 0.5 M H2SO4 in comparison with pure Ru x Se and MoS2, which requires an overpotential of 120 mV to deliver a 10 mA cm-2 current density and gives a Tafel slope of 72.2 mV dec-1. In addition, the hybrid electrocatalyst also exhibits superior electrochemical stability during the long-term HER process in both acidic media and alkaline media.