RESUMO
AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.
RESUMO
Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to â¼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.
Assuntos
Inativação Gênica , Interferência de RNA , RNA Mensageiro , Animais , Humanos , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
A nonlinear Tomlinson-Harashima precoding (NTHP) scheme has been verified for its capability to effectively address both the linear and nonlinear inter-symbol interferences (ISIs) arising in the intensity-modulation direct-detection (IM-DD) fiber optics transmission. Nevertheless, the application of the NTHP scheme may significantly increase the number of levels for the intensity modulated signals, resulting in the reduction of both eye width and receiver sensitivity. Here, we propose a fractionally spaced NTHP with a weight clustering (FS-NTHP-WC) scheme. Consequently, an accurate ISI feedback can be obtained to enlarge the eye width; meanwhile a hardware-efficient implementation without the equalization penalty can be achieved by weight clustering and pruning. When the C-band 100â Gbaud/λ PAM-4 signals are transmitted, our proposed FS-NTHP-WC scheme not only can achieve 0.25â dB and 0.5â dB gains of receiver sensitivity under back-to-back (B2B) and 2-km standard single-mode fiber (SSMF) transmission conditions, respectively, but can also cut down the computational complexity by 90% and 76% in terms of the number of multiplications and additions, respectively, in comparison with the NTHP scheme.
RESUMO
Condensation of 3,3'-diamino-2,2'-ethylene-bridged azobenzene with 1,2,4,5-tetrakis-(4-formylphenyl) benzene produces a visible light responsive porous 2D covalent organic framework, COF-bAzo-TFPB, with a large surface area, good crystallinity, and thermal and chemical stability. The results demonstrate that the elaborated designed linker can make azo unit on the COF-bAzo-TFPB skeleton undergo reversible photoisomerization. This work expands the application scope of covalent organic frameworks in photo-controlled release, uptake of guest molecules, dynamic photoswitching, and UV-sensitive functions.
Assuntos
Estruturas Metalorgânicas , Compostos Azo , Benzeno , LuzRESUMO
Although recent regulatory approval of splice-switching oligonucleotides (SSOs) for the treatment of neuromuscular disease such as Duchenne muscular dystrophy has been an advance for the splice-switching field, current SSO chemistries have shown limited clinical benefit due to poor pharmacology. To overcome limitations of existing technologies, we engineered chimeric stereopure oligonucleotides with phosphorothioate (PS) and phosphoryl guanidine-containing (PN) backbones. We demonstrate that these chimeric stereopure oligonucleotides have markedly improved pharmacology and efficacy compared with PS-modified oligonucleotides, preventing premature death and improving median survival from 49 days to at least 280 days in a dystrophic mouse model with an aggressive phenotype. These data demonstrate that chemical optimization alone can profoundly impact oligonucleotide pharmacology and highlight the potential for continued innovation around the oligonucleotide backbone. More specifically, we conclude that chimeric stereopure oligonucleotides are a promising splice-switching modality with potential for the treatment of neuromuscular and other genetic diseases impacting difficult to reach tissues such as the skeletal muscle and heart.
Assuntos
Distrofia Muscular de Duchenne , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Animais , Éxons , Camundongos , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Splicing de RNA/efeitos dos fármacosRESUMO
Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.
In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.
Assuntos
Oligonucleotídeos Antissenso , Animais , Células Cultivadas , Sistema Nervoso Central , Guanidina/química , Camundongos , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos , Ribonuclease H/metabolismoRESUMO
Cadmium (Cd), a widely distributed environmental pollutant in agroecosystems, causes negative effects on crops and herbivores through bottom-up processes. The gut microbial community of an insect can play a critical role in response to metal stress. To understand how microbiota affect the stress responses of organisms to heavy metals in agroecosystems, we initially used 16S rRNA sequencing to characterize the larval gut microbiota of Chilo suppressalis, an important agricultural pest, exposed to a diet containing Cd. The species richness, diversity, and composition of the gut microbial community was then analyzed. Results revealed that while the richness (Chao1 and ACE) of gut microbiota in larvae exposed to Cd was not significantly affected, diversity (Shannon and Simpson) was reduced due to changes in species distribution and relative abundance. Overall, the most abundant genus was Enterococcus, while the abundance of the genera Micrococcaceae and Faecalibaculum in the control significantly superior to that in Cd-exposed pests. Phylogenetic investigation of microbial communities by the reconstruction of unobserved states (PICRUSt) showed that the intestinal microorganisms appear to participate in 34 pathways, especially those used in environmental information processing and the metabolism of the organism. This study suggests that the gut microbiota of C. suppressalis are significantly impacted by Cd exposure and highlights the importance of the gut microbiome in host stress responses and negative effects of Cd pollution in agroecosystems.
RESUMO
Eicosapentaenoic acid (EPA) is a vital É·-3 polyunsaturated fatty acid (PUFA) for human body with various physiological functions. In this study, we proposed an adaptive evolutionary strategy based on high-temperature and high-oxygen two-factor stress to increase the EPA production capacity of Schizochytrium. High-temperature stress was used to increase EPA yield, and high oxygen was implemented to continuously stimulate cell growth and lipid accumulation. The biomass and EPA production of ALE-D50 reached 35.33 g/L and 1.54 g/L, which were 43.85% and 71.11% higher than that of the original strain, respectively. Lower in vivo reactive oxygen species levels indicated that the evolved strain possessed stronger antioxidant activity. Liquid chromatography-mass spectrometry metabolomics showed that enhanced glucose consumption and glycolysis metabolism, as well as a weakened tricarboxylic acid cycle and reduced amino acid metabolic tributaries in the evolved strain, might be associated with increased growth and EPA synthesis. Finally, the lipid production and EPA production in a fed-batch fermentation were further increased to 48.93 g/L and 3.55 g/L, improving by 54.30% and 90.86%, respectively. This study provides a novel pathway for promoting EPA biosynthesis in Schizochytrium.
Assuntos
Ácido Eicosapentaenoico , Metabolômica , Estramenópilas , Ácido Eicosapentaenoico/biossíntese , Ácido Eicosapentaenoico/metabolismo , Estramenópilas/metabolismo , Estramenópilas/crescimento & desenvolvimento , Estresse Fisiológico , Adaptação FisiológicaRESUMO
Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.
Assuntos
Aminoácidos , Biofilmes , Escherichia coli/genética , Perfilação da Expressão Gênica , Trifosfato de AdenosinaRESUMO
Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more ß turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.
Assuntos
Acidithiobacillus , Proteínas de Bactérias , Extremófilos , Transativadores , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cobre/metabolismo , Extremófilos/genética , Extremófilos/metabolismo , Íons/metabolismo , Metais/metabolismo , Ligação Proteica , Transativadores/genética , Transativadores/metabolismoRESUMO
A Volterra nonlinear equalizer (VNLE) can effectively mitigate both linear and nonlinear impairments arising in intensity-modulation direct-detection (IM-DD) transmission systems. However, the high computational complexity of the VNLE hinders its applications. Here, we propose a hardware-efficient Volterra equalizer with weight-sharing pruning (VE-WSP). Such an equalizer first uses the k-means++ clustering algorithm for the weight sharing within the same cluster, and then ranks the cluster centroid weight for pruning, leading to a significant computational complexity reduction without sacrificing any equalization performance. We experimentally verified that the use of VE-WSP can enable the C-band 100-GBaud/λ PAM-4 transmission over a 1-km standard single-mode fiber (SSMF), to reach the 20% soft-decision forward error correction (SD-FEC) threshold. Meanwhile, the proposed VE-WSP can reduce the computational complexity by 51% and 21%, in terms of multiplication and addition, respectively, in comparison with the VNLE.
RESUMO
Transmitter dispersion eye closure quaternary (TDECQ) is a vital metric to characterize the quality of four-level pulse amplitude modulation (PAM-4) optical signals. However, the traditional TDECQ assessment scheme is complex and time consuming, with heavy iterative operations. Therefore, accelerating the TDECQ assessment has great significance for photonic data-center interconnection (DCI) applications. Here, we propose and experimentally demonstrate a TDECQ assessment based on linear-convolutional neural network (L-CNN) with the 1 × 1 convolutional kernel to reduce the implementation complexity. Our experimental results verify that the lightweight L-CNN can realize the accurate TDECQ assessment, without the involvement of nonlinear activation functions (NAFs). The mean absolute error (MAE) of 26.5625 and 53.125â GBaud PAM-4 signals are 0.16â dB and 0.18â dB, respectively, over a TDECQ range from 1.5 to 4.0â dB. Meanwhile, in comparison with existing CNN-based schemes, the L-CNN based TDECQ assessment scheme only needs 2048 multiplications, which have been reduced by five orders of magnitude.
RESUMO
Molecular chaperone CbpA from extreme acidophile Acidithiobacillus caldus was applied to improve acid tolerance of Escherichia coli via CRISPR/Cas9. Cell growth and viability of plasmid complementary strain indicated the importance of cbpAAc for bacteria acid tolerance. With in situ gene replacement by CRISPR/Cas9 system, colony formation unit (CFU) of genome recombinant strain BL21-ΔcbpA/AccbpA showed 7.7 times higher cell viability than deficient strain BL21-ΔcbpA and 2.3 times higher than wild type. Cell morphology observation using Field Emission Scanning Electron Microscopy (FESEM) revealed cell breakage of BL21-ΔcbpA and significant recovery of BL21-ΔcbpA/AccbpA. The intracellular ATP level of all strains gradually decreased along with the increased stress time. Particularly, the value of recombinant strain was 56.0% lower than that of deficient strain after 5 h, indicating that the recombinant strain consumed a lot of energy to resist acid stress. The arginine concentration in BL21-ΔcbpA/AccbpA was double that of BL21-ΔcbpA, while the aspartate and glutamate contents were 14.8% and 6.2% higher, respectively, compared to that of wild type. Moreover, RNA-Seq analysis examined 93 genes down-regulated in BL21-ΔcbpA compared to wild type strain, while 123 genes were up-regulated in BL21-ΔcbpA/AccbpA compared to BL21-ΔcbpA, with an emphasis on energy metabolism, transport, and cell components. Finally, the working model in response to acid stress of cbpA from A. caldus was developed. This study constructed a recombinant strain resistant to acid stress and also provided a reference for enhancing microorganisms' robustness to various conditions.
Assuntos
Escherichia coli , Extremófilos , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos , Ácidos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
The number of base stations (BSs) for the fifth generation (5G) wireless network is substantially increased, as each coverage is greatly reduced. Therefore, both the miniaturization and the densification of BSs suffer from the challenges of electrical power supply and deployment cost. Here, we present an optically powered 5G fronthaul network, in support of the co-propagation of spatial-division-multiplexing (SDM) energy light and wavelength-division-multiplexing (WDM) 5G new radio (NR) signals over the weakly-coupled multicore fiber (WC-MCF). When the 60-W energy light at 1064.8-nm is equally distributed among the outer six cores, and the 9-Gbit/s 5G NR WDM signals are transmitted over the central core of 1-km WC seven-core fiber (WC-7CF), we can collect total 11.9-W electrical power at the remote node, for the purpose of optically powered small cells. Meanwhile, the error-vector magnitude (EVM) values of 1.5-Gbit/s 5G NR 64-level quadrature amplitude modulation orthogonal frequency division multiplexing (64QAM-OFDM) signals at the central frequency of 3.5 GHz fluctuate within a range of 0.3%â¼0.39%, under a received electrical power of -25 dBm, for all six-wavelength channels. Six optically powered small cells are equipped with the characteristics of centralized management and flexible access-rate.
RESUMO
The performance of high baud-rate intensity modulation direct detection (IM-DD) transmissions is severely degraded by both the linear and nonlinear inter-symbol interference (ISI). Here, we propose and experimentally demonstrate a transmitter-side look-up-table pre-distortion combined with nonlinear Tomlinson-Harashima pre-coding (LUT_PD-NTHP) scheme with the capability of mitigating the linear and nonlinear ISI simultaneously, enabling a C-band 200 Gbit/s/λ PAM-4 transmission over 2-km standard single mode fiber (SSMF), under an end-to-end 10-dB bandwidth of about 20 GHz. The proposed LUT_PD-NTHP scheme is experimentally verified to be superior to the LUT pre-distortion combined with linear THP (LUT_PD-LTHP) scheme, in terms of both the receiver sensitivity and the LUT storage requirement, when only the feed-forward equalization (FFE) is used at the receiver-side. In particular, after the 200 Gbit/s PAM-4 signal transmission over the 2-km SSMF without the chromatic dispersion (CD) compensation, the proposed LUT_PD-NTHP scheme with a LUT pattern length of 3 possesses not only 0.25 dB improvement of the receiver sensitivity but also about 99% LUT pattern reduction, in comparison with the LUT_PD-LTHP scheme with a LUT pattern length of 5.
RESUMO
The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsRAc) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsRAc monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsRAc protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsRAc could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsRAc negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsRAc did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: ⢠Identification of a MarR family EPS-associated transcriptional regulator, named EpsRAc. ⢠Cu(I) can bind to the EpsRAc protein with low affinity. ⢠EpsRAc negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.
Assuntos
Acidithiobacillus , Proteínas de Escherichia coli , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Cobre/metabolismo , Cobre/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Reynolds averaged N-S equation and dynamic equation for nanoparticles are numerically solved in the two-phase flow around cylinders, and the distributions of the concentration M0 and geometric mean diameter dg of particles are given. Some of the results are validated by comparing with previous results. The effects of particle coagulation and breakage and the initial particle concentration m00 and size d0 on the particle distribution are analyzed. The results show that for the flow around a single cylinder, M0 is reduced along the flow direction. Placing a cylinder in a uniform flow will promote particle breakage. For the flow around multiple cylinders, the values of M0 behind the cylinders oscillate along the spanwise direction, and the wake region in the flow direction is shorter than that for the flow around a single cylinder. For the initial monodisperse particles, the values of dg increase along the flow direction and the effect of particle coagulation is larger than that of particle breakage. The values of dg fluctuate along the spanwise direction; the closer to the cylinders, the more frequent the fluctuations of dg values. For the initial polydisperse particles with d0 = 98 nm and geometric standard deviation σ = 1.65, the variations of dg values along the flow and spanwise directions show the same trend as for the initial monodisperse particles, although the differences are that the values of dg are almost the same for the cases with and without considering particle breakage, while the distribution of dg along the spanwise direction is flatter in the case with initial polydisperse particles.
RESUMO
The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.
Assuntos
Acidithiobacillus/metabolismo , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Proteínas Repressoras/metabolismo , Acidithiobacillus/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/genéticaRESUMO
Efficient co-transmission of 10-W power light and optically carried 5G new radio (NR) signal over standard single-mode fiber (SSMF) is experimentally demonstrated, with an optical power transmission efficiency (OPTE) of power light up to 71.8%. This efficiency record is enabled by carefully manipulating the linewidth of the power light and appropriately adjusting the wavelength spacing between the power light and 5G NR optical signal to effectively mitigate the nonlinear effect arising in the SSMF. In the experiment, the optically carried 5G NR 64-level quadrature amplitude modulation orthogonal frequency division multiplexing signal at 1550 nm, with a data rate of 1.5 Gbit/s, is successfully co-propagated with 10-W power light at 1064 nm over 1-km SSMF. The error-vector magnitude (EVM) is 0.48% under a received electrical power of -25dBm. In comparison with back-to-back transmission, only slight EVM degradation of 0.02% is observed, showing that the 5G NR optical signal is almost unaffected by the existence of power light. Moreover, the power fluctuation of the collected power light is less than 0.2% over 6 h, while the EVM fluctuation is smaller than 0.01% within 30 min. Our scheme is promising to realize an optically powered remote antenna unit through the existing 5G fronthaul SSMF link.
RESUMO
To understand the acid-resistant mechanism of bioleaching microorganism Acidithiobacillus caldus CCTCC M 2018054, its physiology and metabolic changes at the transcriptional level under extreme acid stress were systemically studied. Scanning electron microscopy (SEM), Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) showed that with an increase in acidity, the absorption peak of sulfur oxidation-related functional groups such as S-O decreased significantly, and a dense sulfur passivation film appeared on the surface of the ore. Confocal laser scanning microscopy (CLSM) revealed that coverage scale of extracellular polymeric substance (EPS) and biofilm fluctuated accordingly along with the increasing acid stress (pH-stat 1.5, 1.2 0.9 and 0.6) during the bioleaching process. In response to acid stress, the increased levels of intracellular glutamic acid, alanine, cysteine, and proline contributed to the maintenance of intracellular pH homeostasis via decarboxylation and alkaline neutralization. Higher unsaturated fatty acid content was closely related to membrane fluidity. Up to 490 and 447 differentially expressed genes (DEGs) were identified at pH 1.5 vs pH 1.2 and pH 1.2 vs pH 0.9, respectively, and 177 common DEGs were associated with two-component system (TCS) regulation, transporter regulation, energy metabolism, and stress response. The upregulation of kdpB helped cells defend against proton invasion, whereas the downregulation of cysB and cbl implied stronger oxidation of sulfur compounds. The transcriptional level of sqr, sor, and soxA was significantly increased and consolidated the energy supply needed for resisting acid stress. Furthermore, eight of the identified DEGs (sor, cbl, ompA, atpF, nuoH, nuoC, sqr, grxB) were verified as being related to the acid stress response process. This study contributes toward expanding the application of these acidophiles in industrial bioleaching.