Mn-Anti-CTLA4-CREKA-Sericin Nanotheragnostics for Enhanced Magnetic Resonance Imaging and Tumor Immunotherapy.

The integration of magnetic resonance imaging (MRI), cGAS-STING, and anti-CTLA-4 (aCTLA-4) based immunotherapy offers new opportunities for tumor precision therapy. However, the precise delivery of aCTLA-4 and manganese (Mn), an activator of cGAS, to tumors remains a major challenge for enhanced MRI and active immunotherapy. Herein, a theragnostic nanosphere Mn-CREKA-aCTLA-4-SS (MCCS) is prepared by covalently assembling Mn2+, silk sericin (SS), pentapeptide CREKA, and aCTLA-4. MCCS are stable with an average size of 160 nm and is almost negatively charged or neutral at pH 5.5/7.4. T1-weighted images showed MCCS actively targeted tumors to improve the relaxation rate r1 and contrast time of MRI. This studies demonstrated MCCS raises reactive oxygen species levels, activates the cGAS-STING pathway, stimulates effectors CD8+ and CD80+ T cells, reduces regulatory T cell numbers, and increases IFN-γ and granzyme secretion, thereby inducing tumor cells autophagy and apoptosis in vitro and in vivo. Also, MCCS are biocompatible and biosafe. These studies show the great potential of Mn-/SS-based integrative material MCCS for precision and personalized tumor nanotheragnostics.

[Research progress in pharmacological effects of Erigeron breviscapus and its active ingredients for treatment of Alzheimer's disease].

Alzheimer's disease(AD) is a neurodegenerative disease that has no effective drug to cure it. Studies in several AD models have shown that Erigeron breviscapus and its active ingredients(scutellarin and caffeoylquinic acid) could improve/enhance the learning and memory ability, and the mechanisms are associated with inhibiting amyloid ß(Aß) production, aggregation, fibrosis and Aß neurotoxicity toxicity, regulating cholinergic nervous system, inhibiting oxidative stress and inflammation, inhibiting tau hyperphosphorylation, improving mitochondrial function, and resisting neuronal apoptosis. This article systematically reviewed the research progress of E. breviscapus and its active ingredients for treatment of AD in AD models, in the expectation of providing references for further development of E. breviscapus's medicinal potential.

Characterization of the N-methyltransferases involved in the biosynthesis of toxoflavin, fervenulin and reumycin from Streptomyces hiroshimensis ATCC53615.

Toxoflavin (1), fervenulin (2), and reumycin (3), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N-methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4-e]-as-triazine-5,7(6H,8H)-dione (4) to generate 1. However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N-methyltransferases required for the biosynthesis of 1 and 2, we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N-methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N-methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1.

Air-stable self-powered photodetector based on TaSe2/WS2/TaSe2 asymmetric heterojunction with surface self-passivation.

Two-dimensional (2D) transition metal dichalcogenides are highly suitable for constructing junction photodetectors because of their suspended bond-free surface and adjustable bandgap. Additional stable layers are often used to ensure the stability of photodetectors. Unfortunately, they often increase the complexity of preparation and cause performance degradation of devices. Considering the self-passivation behavior of TaSe2, we designed and fabricated a novel self-powered TaSe2/WS2/TaSe2 asymmetric heterojunction photodetector. The heterojunction photodetector shows excellent photoelectric performance and photovoltaic characteristics, achieving a high responsivity of 292 mA/W, an excellent specific detectivity of 2.43 × 1011 Jones, a considerable external quantum efficiency of 57 %, a large optical switching ratio of 2.6 × 105, a fast rise/decay time of 43/54 µs, a high open-circuit voltage of 0.23 V, and a short-circuit current of 2.28 nA under 633 nm laser irradiation at zero bias. Moreover, the device also shows a favorable optical response to 488 and 532 nm lasers. Notably, it exhibits excellent environmental long-term stability with the performance only decreasing âˆ¼ 5.6 % after exposed to air for 3 months. This study provides a strategy for the development of air-stable self-powered photodetectors based on 2D materials.

Controlled growth of 3R phase niobium diselenide and its properties.

Inversion symmetry broken 3R phase transition metal dichalcogenides (TMDs) show fascinating prospects in spintronics, valleytronics, and nonlinear optics. However, the controlled synthesis of 3R phase TMDs is still a great challenge. In this work, two-dimensional 3R-NbSe2 single crystals up to 0.2 mm were synthesized for the first time through chemical vapor deposition method by designing a space-confined system. The crystal size and morphology can be controlled by the location of the stacked substrates and the amount of the Nb2O5 precursor. Scanning transmission electron microscopy and Raman measurements reveal the NbSe2 exhibits a pure 3R stacking mode with relatively weak interlayer van der Waals interactions. Importantly, 3R-NbSe2 shows obvious second harmonic generation signal which intensity intensified as thickness increases. Density functional theory calculations and optical absorption demonstrate the coexistence of metallic and semiconducting optical properties of 3R-NbSe2. We designed a NbSe2/WS2/NbSe2 photodetector utilizing the metallicity of 3R-NbSe2, which shows good performance especially an ultrafast response (6-7 µs, 0.5 ms - 7.9 s for Au electrodes in literature). The proposed strategy and findings are of great significance for the growth of many other 3R-TMDs and applications of nonlinear optical and ultrafast devices.

The impact of sarcopenia on the incidence of postoperative outcomes following spine surgery: Systematic review and meta-analysis.

PURPOSE: Sarcopenia is considered to be an important predictor of adverse outcomes following spinal surgery, but the specific relationship between the two is not clear. The purpose of this meta-analysis is to systematically review all relevant studies to evaluate the impact of sarcopenia on spinal surgery outcomes. METHODS: We systematically searched PubMed, Embase and the Cochrane Library for relevant articles published on or before January 9, 2023. The pooled odds ratio (OR) with 95% confidence intervals (CIs) was calculated in a random effects meta-analysis. The main outcome was the risk of adverse outcomes after spinal surgery, including adverse events and mortality. This systematic review and meta-analysis was conducted following the PRISMA guidelines to evaluate the impact of sarcopenia on spinal surgery outcomes. In addition, we also conducted a subgroup analysis and leave-one-out sensitivity analyses to explore the main sources of heterogeneity and the stability of the results. RESULTS: Twenty-four cohort studies, with a total of 243,453 participants, met the inclusion criteria. The meta-analysis showed that sarcopenia was significantly associated with adverse events (OR 1.63, 95% CI 1.17-2.27, P < 0.001) but was no significantly associated with mortality (OR 1.17, 95% CI 0.93-1.46, P = 0.180), infection (OR 2.24, 95% CI 0.95-5.26, P < 0.001), 30-day reoperation (OR 1.47, 95% CI 0.92-2.36, P = 0.413), deep vein thrombosis (OR 1.78, 95% CI 0.69-4.61, P = 0.234), postoperative home discharge (OR 0.60, 95% CI 0.26-1.37, P = 0.002) and blood transfusion (OR 3.28, 95% CI 0.74-14.64, P = 0.015). CONCLUSION: The current meta-analysis showed that patients with sarcopenia have an increased risk of adverse events and mortality after spinal surgery. However, these results must be carefully interpreted because the number of studies included is small and the studies are significantly different. These findings may help to increase the clinicians' awareness of the risks concerning patients with sarcopenia to improve their prognosis.

Restoration of aquaporin-4 polarization in the spinal glymphatic system by metformin in rats with painful diabetic neuropathy.

Painful diabetic neuropathy (PDN) is a common complication in patients with diabetes, and its underlying mechanism remains unclear. Aquaporin-4 (AQP4) plays a crucial role in removing metabolic waste in the glymphatic system. In this study, we aimed to explore the relationship between the spinal glymphatic system and the effect of metformin on PDN. Male Sprague-Dawley rats were randomly allocated into the control group ( n = 10), the PDN group ( n = 10), and the metformin group ( n = 10). A high-fat and high-glucose diet combined with low-dose streptozotocin was used to induce PDN rats. We detected the clearance rate of the contrast agent in the spinal cord of each rat by MRI to reflect the function of the glymphatic system. Immunofluorescence was used to detect the localization of perivascular AQP4 in astrocyte endfeet. Furthermore, we measured the expression of AQP4 in the spinal cord by Western blot. Compared with the rats in the control group, PDN rats exhibited enhanced mechanical allodynia, decreased clearance rate of the contrast agent in the spinal glymphatic system, reversed AQP4 polarization, and increased expression of AQP4. After being treated with metformin, the rats showed opposite changes in the above characteristics. The analgesic effect of metformin on PDN may be related to its ability to restore spinal AQP4 polarization, thus promoting the function of the spinal glymphatic system.

Self-Driven Gr/WSe2/Gr Photodetector with High Performance Based on Asymmetric Schottky van der Waals Contacts.

Two-dimensional (2D) self-driven photodetectors have a wide range of applications in wearable, imaging, and flexible electronics. However, the preparation of most self-powered photodetectors is still complex and time-consuming. Simultaneously, the constant work function of a metal, numerous defects, and a large Schottky barrier at the 2D/metal interface hinder the transmission and collection of optical carriers, which will suppress the optical responsivity of the device. This paper proposed a self-driven graphene/WSe2/graphene (Gr/WSe2/Gr) photodetector with asymmetric Schottky van der Waals (vdWs) contacts. The vdWs contacts are formed by transferring Gr as electrodes using the dry-transfer method, obviating the limitations of defects and Fermi-level pinning at the interface of electrodes made by conventional metal deposition methods to a great extent and resulting in superior dynamic response, which leads to a more efficient and faster collection of photogenerated carriers. This work also demonstrates that the significant surface potential difference of Gr electrodes is a crucial factor to ensure their superior performance. The self-driven Gr/WSe2/Gr photodetector exhibits an ultrahigh Ilight/Idark ratio of 106 with a responsivity value of 20.31 mA/W and an open-circuit voltage of 0.37 V at zero bias. The photodetector also has ultrafast response speeds of 42.9 and 56.0 µs. This paper provides a feasible way to develop self-driven optoelectronic devices with a simple structure and excellent performance.

Surface-Enhanced Raman Spectroscopy for Boosting Electrochemical CO2 Reduction on Amorphous-Surfaced Tin Oxide Supported by MXene.

Amorphous materials disrupt the intrinsic linear scalar dependence seen in their crystalline counterparts, typically exhibiting enhanced catalytic characteristics. Nevertheless, substantial obstacles remain in terms of boosting their stability, enhancing their conductivity, and elucidating distinct catalytic mechanisms. Herein, a core-shell catalyst, comprising a crystalline SnO2 core and an amorphous SnOx shell supported on MXene (denoted as SnO2@SnOx/MXene), was prepared utilizing hydrothermal and solution reduction methods. The SnO2@SnOx/MXene catalyst excels in the electrocatalytic conversion of CO2 to formate, yielding a Faradaic efficiency (FE) as high as 93% for formate production at -1.17 V vs RHE and demonstrating exceptional durability. Both density functional theory (DFT) calculations and experimental results indicate that the SnOx shell bolsters formate formation by fine-tuning the adsorption energy of the *OCHO intermediate. In SnO2@SnOx/MXene, MXene plays a vital role in enhancing the conductivity and stability of the amorphous shell and especially amplifying Raman signals of catalyst components. The ex/in situ surface-enhanced Raman scattering (SERS) application further confirms the formation of amorphous SnOx and further enables the direct detection of the formation of the intermediate species. This work provides the basis for the application of amorphous materials in practical electrocatalytic reduction of CO2 reduction.

Culture-independent methods for studying environmental microorganisms: methods, application, and perspective.

Since the application of molecular methods, culture-independent methods (CIMs) have been developed to study microbial communities from various environments. In the past 20 years, several methods based on the direct amplification and analyses of the small subunit ribosomal RNA gene have been developed to directly study environmental microorganisms. These methods include denaturing/temperature gradient gel electrophoresis, single-strand-conformation polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridization have also been adopted. In recent years, several novel fields of investigation such as metagenomics, metatranscriptomics, metaproteomics, and single-cell genomics were developed, largely propelled by the innovation and application of next-generation sequencing methods. Several single-cell-based technologies such as Raman microspectroscopy and nano-scale secondary ion mass spectrometry are also increasingly used in the fields of microbial ecology and environmental microbiology. The application of these methods has revolutionized microbiology by allowing scientists to directly analyze natural microbial communities in situ, including their genes, transcripts, proteins, and metabolites and how their interactions impact their distribution patterns. In this review, we present an up-to-date review on different CIMs and their applications, our focuses are on the comparison of different CIMs and their application in the analyses of microbial diversities and communities.

ß-Hydroxybutyrate Attenuates Painful Diabetic Neuropathy via Restoration of the Aquaporin-4 Polarity in the Spinal Glymphatic System.

Waste removal is essential for maintaining homeostasis and the normal function of the central nervous system (CNS). The glymphatic system based on aquaporin-4 (AQP4) water channels on the endfeet of astrocytes is recently discovered as the excretion pathway for metabolic waste products of CNS. In the CNS, α-syntrophin (SNTA1) directly or indirectly anchors AQP4 in astrocyte membranes facing blood vessels. Studies have indicated that ß-hydroxybutyrate (BHB) can raise the expression of SNTA1 and thus restoring AQP4 polarity in mice models with Alzheimer's disease. The study aims to evaluate the neuroprotective mechanism of BHB in rats with painful diabetic neuropathy (PDN). PDN rats were modeled under a high-fat and high-glucose diet with a low dose of streptozotocin. Magnetic resonance imaging (MRI) was applied to observe the clearance of contrast to indicate the functional variability of the spinal glymphatic system. Mechanical allodynia was assessed by paw withdrawal threshold. The expressions of SNTA1 and AQP4 were tested, and the polarity reversal of AQP4 protein was measured. As demonstrated, PDN rats were manifested with deceased contrast clearance of the spinal glymphatic system, enhanced mechanical allodynia, lower expression of SNTA1, higher expression of AQP4, and reversed polarity of AQP4 protein. An opposite change in the above characteristics was observed in rats being treated with BHB. This is the first study that demonstrated the neuroprotective mechanism of BHB to attenuate PDN via restoration of the AQP4 polarity in the spinal glymphatic system and provides a promising therapeutic strategy for PDN.

Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis.

Microorganisms play important roles in the tobacco aging process. However, microbial communities on flue-cured tobacco leaves (FCTL) remain largely unknown. In this study, the total microbial genomic DNA of unaged and aging FCTL from Zimbabwe were isolated using a culture-independent method, and the bacterial communities were investigated through analyzing two 16S rRNA gene libraries. Eighty-four and 65 operational taxonomic units were obtained from the libraries of the unaged and aging FCTL, respectively. The following genera were represented more than 4% in both libraries (aging and unaged library): Sphingomonas (4.84%, 4.18%), Stenotrophomonas (4.84%, 5.23%), Erwinia (5.81%, 4.88%), Pantoea (19.35%, 18.47%), and Pseudomonas (21.29%, 24.04%). The dominant species varied between the two libraries. Specifically, several dominant species in unaged FCTL including Pseudomonas fulva, Pseudomonas sp. (AM909658), Klebsiella sp. (HM584796), and Pantoea sp. (AY501386) were not identified in aging FCTL, while several dominant species in aging FCTL such as Pantoea sp. (GU566350), Pseudomonas sp. (EF157292), and Buttiauxella izardii were not found in unaged FCTL. The phylogenetic analysis showed that bacteria from unaged and aging FCTL were divided into two clades, and two unique subclades were identified in aging FCTL. Our results revealed for the first time the bacterial diversities on Zimbabwe tobacco, and provided a basis for clarifying the roles of bacteria in aging process of FCTL.

Bacterial diversities on unaged and aging flue-cured tobacco leaves estimated by 16S rRNA sequence analysis.

Flue-cured tobacco leaves (FCTL) contain abundant bacteria, and these bacteria play very important roles in the tobacco aging process. However, bacterial communities on aging FCTL are not fully understood. In this study, the total microbial genome DNA of unaged and aging flue-cured tobacco K326 were isolated using a culture-independent method, and the bacterial communities were investigated by restriction fragment length polymorphism analysis. Comparison of the number of operational taxonomic units (OTUs) between the cloned libraries from the unaged and aging FCTL showed that the microbial communities between the two groups were different. Fifty and 42 OTUs were obtained from 300 positive clones in unaged and aging FCTL, respectively. Twenty-seven species of bacteria exist in both the unaged and aging FCTL, Bacillus spp. and Pseudomonas spp. were two dominant genera in FCTL. However, 23 bacterial species were only identified from the unaged FCTL, while 15 species were only identified from the aging FCTL. Interestingly, more uncultured bacteria species were found in aging FCTL than in unaged FCTL.

Characterization of Streptomyces piniterrae sp. nov. and Identification of the Putative Gene Cluster Encoding the Biosynthesis of Heliquinomycins.

A novel actinomycete producing heliquinomycin and 9'-methoxy-heliquinomycin, designated strain jys28T, was isolated from rhizosphere soil of Pinus yunnanensis and characterized using a polyphasic approach. The strain had morphological characteristics and chemotaxonomic properties identical to those of members of the genus Streptomyces. It formed spiral chains of spores with spiny surfaces. The menaquinones detected were MK-9(H6), MK-9(H8) and MK-9(H4). The major fatty acids were iso-C16:0, C15:0, C16:1ω7с and anteiso-C15:0. The phospholipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine and phosphatidylinositol mannoside. The DNA G + C content of the draft genome sequence, consisting of 8.5 Mbp, was 70.6%. Analysis of the 16S rRNA gene sequence showed that strain jys28T belongs to the genus Streptomyces with the highest sequence similarities to Streptomyces chattanoogensis NBRC 13058T (99.2%) and Streptomyces lydicus DSM 40002T (99.2%) and phylogenetically clustered with them. Multilocus sequence analysis based on five other house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and the low level of DNA-DNA relatedness and phenotypic differences allowed the novel isolate to be differentiated from its most closely related strains. Therefore, the strain is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces piniterrae sp. nov. is proposed. Furthermore, the putative biosynthetic gene cluster of heliquinomycins was identified and the biosynthetic pathway was discussed. The type strain is jys28T (=CCTCC AA 2018051T =DSM 109823T).

Microwave Absorption Properties of CoS2 Nanocrystals Embedded into Reduced Graphene Oxide.

CoS2 nanoparticles and CoS2/reduced graphene oxide (CoS2/rGO) nanohybrids were fabricated by a unique single-mode microwave-assisted hydrothermal method. The microwave absorption properties of CoS2/rGO composites with different rGO proportions were investigated. Morphology analysis indicated that the CoS2 nanoparticles were uniformly embedded into rGO without aggregation. The complex permittivity of CoS2/rGO nanohybrids could be artificially tuned with the rGO proportions. For the CoS2/rGO 1:2 composite, the minimum reflection loss (RL) of -56.9 dB was achieved at 10.9 GHz for the thickness of 2.2 mm; meanwhile, the RL exceeding -10 dB were obtained in the frequency range of 9.1-13.2 GHz. Compared with other rGO-based materials, CoS2/rGO composite exhibited superior microwave absorption ability at a rather thin thickness and it has great potential to be used as a high-efficiency and tunable microwave absorber.

Fabrication of NiCo2-Anchored Graphene Nanosheets by Liquid-Phase Exfoliation for Excellent Microwave Absorbers.

Graphene nanosheets (GNSs) were prepared by an efficient liquid-phase exfoliation method, and then the NiCo2/GNS nanohybrids were fabricated using the single-mode microwave-assisted hydrothermal technique. The NiCo2/GNS composites with different GNS proportions were investigated as microwave absorbers. Morphology investigation suggested that NiCo2 nanocrystals were uniformly anchored on the GNS without aggregation. The electromagnetic parameters of NiCo2/GNS nanohybrids could be artificially adjusted by changing the GNS proportion, which led to an exceptional microwave-absorbing performance. A reflection loss (RL) exceeding -20 dB was obtained in the frequency range of 5.3-16.4 GHz for the absorber thicknesses of 1.2-3.2 mm, while an optimal RL of -30 dB was achieved at 11.7 GHz for a thickness of 1.6 mm. The enhanced microwave-absorbing performance indicated that the NiCo2/10 wt % GNS composite has great potential for use as an excellent microwave absorber.

A distributed parameter model of transmission line transformer for high voltage nanosecond pulse generation.

A transmission line transformer has potential advantages for nanosecond pulse generation including excellent frequency response and no leakage inductance. The wave propagation process in a secondary mode line is indispensable due to an obvious inside transient electromagnetic transition in this scenario. The equivalent model of the transmission line transformer is crucial for predicting the output waveform and evaluating the effects of magnetic cores on output performance. However, traditional lumped parameter models are not sufficient for nanosecond pulse generation due to the natural neglect of wave propagations in secondary mode lines based on a lumped parameter assumption. In this paper, a distributed parameter model of transmission line transformer was established to investigate wave propagation in the secondary mode line and its influential factors through theoretical analysis and experimental verification. The wave propagation discontinuity in the secondary mode line induced by magnetic cores is emphasized. Characteristics of the magnetic core under a nanosecond pulse were obtained by experiments. Distribution and formation of the secondary mode current were determined for revealing essential wave propagation processes in secondary mode lines. The output waveform and efficiency were found to be affected dramatically by wave propagation discontinuity in secondary mode lines induced by magnetic cores. The proposed distributed parameter model was proved more suitable for nanosecond pulse generation in aspects of secondary mode current, output efficiency, and output waveform. In depth, comprehension of underlying mechanisms and a broader view of the working principle of the transmission line transformer for nanosecond pulse generation can be obtained through this research.

Non-enzymatic pyridine ring formation in the biosynthesis of the rubrolone tropolone alkaloids.

The pyridine ring is a potent pharmacophore in alkaloid natural products. Nonetheless, its biosynthetic pathways are poorly understood. Rubrolones A and B are tropolone alkaloid natural products possessing a unique tetra-substituted pyridine moiety. Here, we report the gene cluster and propose a biosynthetic pathway for rubrolones, identifying a key intermediate that accumulates upon inactivation of sugar biosynthetic genes. Critically, this intermediate was converted to the aglycones of rubrolones by non-enzymatic condensation and cyclization with either ammonia or anthranilic acid to generate the respective pyridine rings. We propose that this non-enzymatic reaction occurs via hydrolysis of the key intermediate, which possesses a 1,5-dione moiety as an amine acceptor capable of cyclization. This study suggests that 1,5-dione moieties may represent a general strategy for pyridine ring biosynthesis, and more broadly highlights the utility of non-enzymatic diversification for exploring and expanding natural product chemical space.

Finding potential ligands for PDZ domains by tailfit, a JAVA program.

OBJECTIVE: To deduce all potential ligands undiscovered experimentally by searching all the proteins containing same C-termini, which can bind a certain PDZ domain. METHODS: We developed a JAVA program for searching short exact sequence matches at C-terminus. According to the known C-termini, which PDZ domains recognized experimentally, Swissprot database has been searched by this program for all potential ligands. RESULTS: Some PDZ domains may have more potential ligand proteins, which are undiscovered yet experimentally. These bioinformatic results also provide clues for studying functions of hypothetical proteins and PDZ domains' protein interactions in many different organisms. CONCLUSION: The results may provide useful clues for discovering potential functions of hypothetical proteins and new functions of known proteins.

[Utilizing tabacco genomic DNA to construct nearly random peptide libraries].

We developed a novel method for constructing nearly random peptide library. Genomic DNAs extracted from tissue or cells of large genome species were digested with frequent cutter to produce short DNA fragments. These short fragments can be considered nearly random. Nearly random peptide libraries can be constructed by cloning the short fragments into appropriate expression vectors and transformation into host cells. Genomic DNA from one species can be digested with different restriction enzymes and ligated to different reading frames to produce several different libraries. In this study, we digested tobacco genomic DNA with two enzymes and cloned into three different reading frames to make totally six nearly random peptide libraries.