Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Study on the inhibition activity and mechanism of Tanreqing against Klebsiella pneumoniae biofilm formation in vitro and in vivo.

Objective: To evaluate the antibacterial effect of Tanreqing (TRQ) against K. pneumoniae and its inhibition activity on bacterial biofilm formation in vitro and in vivo, and to explore the mechanism of the inhibitory effects of TRQ on K. pneumoniae biofilm formation. Methods: An in vitro biofilm model of K. pneumoniae was established, and the impact of TRQ on biofilm formation was evaluated using crystal violet staining and scanning electron microscopy (SEM). Furthermore, the clearance effect of TRQ against K. pneumoniae in the biofilm was assessed using the viable plate counting method; q-RT PCR was used to evaluate the inhibitory effect of different concentrations of TRQ on the expression of biofilm-related genes in Klebsiella pneumoniae; The activity of quorum sensing signal molecule AI-2 was detected by Vibrio harveyi bioluminescence assay; Meanwhile, a guinea pig lung infection model of Klebsiella pneumoniae was constructed, and after treated with drugs, pathological analysis of lung tissue and determination of bacterial load in lung tissue were performed. The treatment groups included TRQ group, imipenem(IPM) group, TRQ+IPM group, and sterile saline group as the control. Results: The formation of K. pneumoniae biofilm was significantly inhibited by TRQ in vitro experiments. Furthermore, when combined with IPM, the clearance of K. pneumoniae in the biofilm was notably increased compared to the TRQ group and IPM group alone. q-RT PCR analysis revealed that TRQ down-regulated the expression of genes related to biofilm formation in K. pneumoniae, specifically luxS, wbbm, wzm, and lsrK, and also inhibited the activity of AI-2 molecules in the bacterium. In vivo experiments demonstrated that TRQ effectively treated guinea pig lung infections, resulting in reduced lung inflammation. Additionally, when combined with IPM, there was a significant reduction in the bacterial load in lung tissue. Conclusion: TRQ as a potential therapeutic agent plays a great role in the treatment of K. pneumoniae infections, particularly in combination with conventional antibiotics. And TRQ can enhanced the clearance effect on the bacterium by inhibiting the K. pneumoniae biofilm formation, which provided experimental evidence in support of clinical treatment of TRQ against K. pneumoniae infections.

Development of potent antibody drug conjugates against ICAM1+ cancer cells in preclinical models of cholangiocarcinoma.

As a highly lethal adenocarcinoma of the hepatobiliary system, outcomes for cholangiocarcinoma (CCA) patients remain prominently poor with a 5-year survival of <10% due to the lack of effective treatment modalities. Targeted therapeutics for CCA are limited and surgical resection of CCA frequently suffers from a high recurrence rate. Here we report two effective targeted therapeutics in this preclinical study for CCA. We first performed a quantitative and unbiased screening of cancer-related antigens using comparative flow cytometry in a panel of human CCA cell lines, and identified intercellular adhesion molecule-1 (ICAM1) as a therapeutic target for CCA. After determining that ICAM1 has the ability to efficiently mediate antibody internalization, we constructed two ICAM1 antibody-drug conjugates (ADCs) by conjugating ICAM1 antibodies to different cytotoxic payloads through cleavable chemical linkers. The efficacies of two ICAM1 ADCs have been evaluated in comparison with the first-line chemodrug Gemcitabine in vitro and in vivo, and ICAM1 antibodies coupled with warhead DX-8951 derivative (DXd) or monomethyl auristatin E (MMAE) elicit a potent and consistent tumor attenuation. In summary, this study paves the road for developing a promising targeted therapeutic candidate for clinical treatment of CCA.

ICAM1 antibody drug conjugates exert potent antitumor activity in papillary and anaplastic thyroid carcinoma.

Treatment options for anaplastic thyroid cancer (ATC) and refractory papillary thyroid carcinoma (PTC) are limited and outcomes remain poor. In this study, we determined via bioinformatic expression analyses and immunohistochemistry staining that intercellular adhesion molecule-1(ICAM1) is an attractive target for ATC and PTC. We designed and engineered two ICAM1-directed antibody-drug conjugate (I1-MMAE and I1-DXd), both of which potently and selectively ablate multiple human ATC and PTC cell lines without affecting non-plastic cells in vitro. Furthermore, I1-MMAE and I1-DXd mediated a potent tumor regression in ATC and PTC xenograft models. To develop a precision medicine, we also explored magnetic resonance imaging (MRI) as a non-invasive biomarker detection method to quantitatively map ICAM1 antigen expression in heterogeneous thyroid tumors. Taken together, this study provides a strong rationale for the further development of I1-MMAE and I1-DXd as promising therapeutic candidates to treat advanced PTC and ATC.

Evaluation of Emergency Response Capacity of Urban Pluvial Flooding Public Service Based on Scenario Simulation.

The evaluation of emergency response capability under different pluvial flooding scenarios is an essential approach to improve the emergency response capability of flood disasters. A new evaluation method of emergency response capacity of urban public services is proposed based on urban pluvial flooding scenario simulation. Firstly, inundation area and depth under different pluvial flooding scenarios are simulated based on the SCS-CN model. Following that, space densities of all indicators include inundation area and depth, road network and the emergency public service institutions. Then, the indicator weight is determined by the combined weighting method of entropy weight and coefficient of variation. Finally, the emergency response capacity index (of each pixel) is calculated based on the graph stacking method. Taking Erqi District, Zhengzhou City as an example, the emergency response capacity of public service under different urban flooding scenarios is evaluated. The results show that the spatial distribution difference of public service emergency response capacity in Erqi District, Zhengzhou City is obvious, and with the increase of the precipitation return period, the high value area of public service emergency response capability decreases gradually and the low value area increases gradually. This method takes into account the specific urban flooding scenario and the layout of public service institutions and road networks that have strong practicability. the results of the evaluation can provide a reference for the construction of urban flood emergency response capacity and provide support for emergency decision-making.

Novel development strategies and challenges for anti-Her2 antibody-drug conjugates.

Antibody-drug conjugates (ADCs) combining potent cytotoxicity of small-molecule drugs with the selectivity and excellent pharmacokinetic profile of monoclonal antibody (mAb) are promising therapeutic modalities for a diverse range of cancers. Owing to overexpression in a wide range of tumors, human epidermal growth factor receptor 2 (Her2) is one of the most utilized targeting antigens for ADCs to treat Her2-positive cancers. Owing to the high density of Her2 antigens on the tumor cells and high affinity and high internalization capacity of corresponding antibodies, 56 anti-Her2 ADCs which applied >10 different types of novel payloads had entered preclinical or clinical trials. Seven of 12 Food and Drug Administration (FDA)-approved ADCs including Polivy (2019), Padcev (2019), EnHertu (2019), Trodelvy (2020), Blenrep (2020), Zynlonta (2021), and Tivdak) (2021) have been approved by FDA in the past three years alone, indicating that the maturing of ADC technology brings more productive clinical outcomes. This review, focusing on the anti-Her2 ADCs in clinical trials or on the market, discusses the strategies to select antibody formats, the linkages between linker and mAb, and effective payloads with particular release and action mechanisms for a good clinical outcome.

Comprehensive Analysis and Expression Profiling of PIN, AUX/LAX, and ABCB Auxin Transporter Gene Families in Solanum tuberosum under Phytohormone Stimuli and Abiotic Stresses.

Auxin is the only plant hormone that exhibits transport polarity mediated by three families: auxin resistant (AUX) 1/like AUX1 (LAX) influx carriers, pin-formed (PIN) efflux carriers, and ATP-binding cassette B (ABCB) influx/efflux carriers. Extensive studies about the biological functions of auxin transporter genes have been reported in model plants. Information regarding these genes in potato remains scarce. Here, we conducted a comprehensive analysis of auxin transporter gene families in potato to examine genomic distributions, phylogeny, co-expression analysis, gene structure and subcellular localization, and expression profiling using bioinformatics tools and qRT-PCR analysis. From these analyses, 5 StLAXs, 10 StPINs, and 22 StABCBs were identified in the potato genome and distributed in 10 of 18 gene modules correlating to the development of various tissues. Transient expression experiments indicated that three representative auxin transporters showed plasma membrane localizations. The responsiveness to auxin and auxin transport inhibitors implied their possible roles in mediating intercellular auxin homoeostasis and redistribution. The differential expression under abscisic acid and abiotic stresses indicated their specific adaptive mechanisms regulating tolerance to environmental stimuli. A large number of auxin-responsive and stress-related cis-elements within their promoters could account for their responsiveness to diverse stresses. Our study aimed to understand the biological significance of potato auxin transporters in hormone signaling and tolerance to environmental stresses.

Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

Evolutionary significance of amino acid permease transporters in 17 plants from Chlorophyta to Angiospermae.

BACKGROUND: Nitrogen is an indispensable nutrient for plant growth. It is used and transported in the form of amino acids in living organisms. Transporting amino acids to various parts of plants requires relevant transport proteins, such as amino acid permeases (AAPs), which were our focus in this study. RESULTS: We found that 5 AAP genes were present in Chlorophyte species and more AAP genes were predicted in Bryophyta and Lycophytes. Two main groups were defined and group I comprised 5 clades. Our phylogenetic analysis indicated that the origin of clades 2, 3, and 4 is Gymnospermae and that these clades are closely related. The members of clade 1 included Chlorophyta to Gymnospermae. Group II, as a new branch consisting of non-seed plants, is first proposed in our research. Our results also indicated that the AAP family was already present in Chlorophyta and then expanded accompanying the development of vasculature. Concurrently, the AAP family experienced multiple duplication events that promoted the generation of new functions and differentiation of sub-functions. CONCLUSIONS: Our findings suggest that the AAP gene originated in Chlorophyta, and some non-seed AAP genes clustered in one group. A second group, which contained plants of all evolutionary stages, indicated the evolution of AAPs. These new findings can be used to guide future research.

Genome-Wide Analysis of the Lateral Organ Boundaries Domain (LBD) Gene Family in Solanum tuberosum.

Lateral organ boundaries domain (LBD) proteins belong to a particular class of transcription factors of lateral organ boundary (LOB) specific domains that play essential roles in plant growth and development. However, a potato phylogenetic analysis of the LBD family has not been fully studied by scholars and researchers. In this research, bioinformatics methods and the growth of potatoes were used to identify 43 StLBD proteins. We separated them into seven subfamilies: Ia, Ib, Ic, Id, Ie, IIa and IIb. The number of amino acids encoded by the potato LBD family ranged from 94 to 327. The theoretical isoelectric point distribution ranged from 4.16 to 9.12 Kda, and they were distributed among 10 chromosomes. The results of qRT-PCR showed that the expression levels of StLBD2-6 and StLBD3-5 were up-regulated under drought stress in the stem. The expression levels of StLBD1-5 and StLBD2-6 were down-regulated in leaves. We hypothesized that StLBD1-5 was down-regulated under drought stress, and that StLBD2-6 and StLBD3-5 up-regulation might help to maintain the normal metabolism of potato and enhance the potatoes' resistance to drought.

Characterization of Beta-Lactamases in Bloodstream-Infection Escherichia coli: Dissemination of blaADC - 162 and blaCMY- 2 Among Bacteria via an IncF Plasmid.

OBJECTIVES: To describe the molecular characteristics of beta-lactamases in bloodstream-infection Escherichia coli isolated from elderly patients, and to determine the genotypic patterns of bla CMY - 2 and bla ADC - 162. METHODS: A total of 50 bloodstream-infection E. coli isolates were obtained from patients aged > 50 years at Shanghai Sixth People's Hospital South Campus during 2015-2018. The isolates were subjected to beta-lactamase detection using phenotypic and molecular methods. Beta-lactamase genes were verified by sequencing and the phylogenetic relationships of the isolates were analyzed by multilocus sequence typing (MLST). The transferability of plasmids carrying bla CMY- 2 and bla ADC- 162 genes was verified by conjugation experiments and plasmid replicon typing. RESULTS: Eight beta-lactamase subtypes were detected in 50 isolates of bloodstream-infection E. coli. bla TEM- 1 (21/50) was the most common beta-lactamase gene, followed by bla CTX-M- 14 (8/50), bla OXA- 27 (5/50), bla CTX-M- 27 (3/50), bla CTX-M- 65 (1/50), bla ADC- 162 (1/50), and bla CMY- 2 (1/50). Of these, bla ADC- 162 (ST95-A), and bla CMY- 2 (ST95-B2) have not previously been reported in bloodstream-infection E. coli. In 21 isolates, beta-lactamase genes were located on conjugative plasmids belonging to incompatibility groups FrepB (n = 7), FIA (n = 1), FIC (n = 2), K (n = 8), N (n = 1), and I (n = 1), and bla CTX-M was associated with the common elements ISEcp1, IS903, and IS26, but with special sequences (region V, region Y, and region W) for ISEcp1 in 14 isolates. CONCLUSION: To the best of our knowledge, this study provides the first molecular characterization of beta-lactamase genes in E. coli isolated from the bloodstream in elderly patients. Beta-lactamase genes were detected at a relatively high frequency in elderly patients with bloodstream E. coli infections. Plasmid replicon analysis showed that horizontal dissemination of beta-lactamase genes was mainly mediated by IncK and IncF plasmids, which could encode multidrug resistance genes. The study also provides the first report of ISAba1-bla ADC - 162-tnpA and ISEcp1-bla CTX-M- 14-IS903-bla CMY- 2-blc-sugE in E. coli, and demonstrates IncF plasmid-mediated bla ADC - 162 and bla CMY- 2 gene dissemination among bacteria.

Comparative Analysis of the PIN Auxin Transporter Gene Family in Different Plant Species: A Focus on Structural and Expression Profiling of PINs in Solanum tuberosum.

Plant growth and morphogenesis largely benefit from cell elongation and expansion and are normally regulated by environmental stimuli and endogenous hormones. Auxin, as one of the most significant plant growth regulators, controls various phases of plant growth and development. The PIN-FORMED (PIN) gene family of trans-membrane proteins considered as auxin efflux carriers plays a pivotal role in polar auxin transport and then mediates the growth of different plant tissues. In this study, the phylogenetic relationship and structural compositions of the PIN gene family in 19 plant species covering plant major lineages from algae to angiosperms were identified and analyzed by employing multiple bioinformatics methods. A total of 155 PIN genes were identified in these species and found that representative of the PIN gene family in algae came into existence and rapidly expanded in angiosperms (seed plants). The phylogenetic analysis indicated that the PIN proteins could be divided into 14 distinct clades, and the origin of PIN proteins could be traced back to the common ancestor of green algae. The structural analysis revealed that two putative types (canonical and noncanonical PINs) existed among the PIN proteins according to the length and the composition of the hydrophilic domain of the protein. The expression analysis of the PIN genes exhibited inordinate responsiveness to auxin (IAA) and ABA both in shoots and roots of Solanum tuberosum. While the majority of the StPINs were up-regulated in shoot and down-regulated in root by the two hormones. The majority of PIN genes had one or more putative auxin responses and ABA-inducible response elements in their promoter regions, respectively, implying that these phytohormones regulated the expression of StPIN genes. Our study emphasized the origin and expansion of the PIN gene family and aimed at providing useful insights for further structural and functional exploration of the PIN gene family in the future.

Polymorphisms of Gene Cassette Promoters of the Class 1 Integron in Clinical Proteus Isolates.

OBJECTIVE: To describe the polymorphisms of gene cassette promoters of the class 1 integron in clinical Proteus isolates and their relationship with antibiotic resistance. METHODS: Polymorphisms of the gene cassette promoter in 153 strains of Proteus were analyzed by PCR and nucleotide sequencing. Variable regions of atypical class 1 integrons were detected by inverse PCR and nucleotide sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyze the phylogenetic relationships of class 1 integron-positive clinical Proteus isolates. Representative beta-lactamase genes (bla), including bla TEM,bla SHV,bla CTX-M-1,bla CTX-M-2,bla CTX-M-8,bla CTX-M-9,bla CTX-M-25 and bla OXA-1, and plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrC, qnrD, qnrS, oqxA, oqxB, qepA, and aac(6')-Ib were also screened using PCR and sequence analysis. RESULTS: Fifteen different gene cassette arrays and 20 different gene cassettes were detected in integron-positive strains. Of them, aadB-aadA2 (37/96) was the most common gene cassette array. Two of these gene cassette arrays (estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3) have not previously been reported. Three different Pc-P2 variants (PcS, PcWTGN-10, PcH1) were detected among the 96 Proteus strains, with PcH1 being the most common (49/96). Strains carrying the promoters PcS or PcWTGN-10 were more resistant to sulfamethoxazole, gentamicin and tobramycin than those carrying PcH1. Strains with weak promoter (PcH1) harbored significantly more intra- and extra-integron antibiotic resistance genes than isolates with strong promoter (PcWTGN-10). Further, among 153 isolates, representative beta-lactamase genes were detected in 70 isolates (bla TEM-1, 54; bla OXA-1, 40; bla CTX-M-3, 12; bla CTX-M-14, 12; bla CTX-M-65, 5; bla CTX-M-15, 2) and representative PMQR genes were detected in 87 isolates (qnrA, 6; qnrB, 3; qnrC, 5; qnrD, 46; qnrS, 5; oqxA, 7; aac(6')-Ib, 13; aac(6')-Ib-cr, 32). CONCLUSION: To the best of our knowledge, this study provides the first evidence for polymorphisms of the class 1 integron variable promoter in clinical Proteus isolates, which generally contain relatively strong promoters. Resistance genotypes showed a higher coincidence rate with the drug-resistant phenotype in strong-promoter-containing strains, resulting in an ability to confer strong resistance to antibiotics among host bacteria and a relatively limited ability to capture gene cassettes. Moreover, strains with relatively weak integron promoters can "afford" a heavier "extra-integron antibiotic resistance gene load". Furthermore, the gene cassettes estX, psp and the gene cassette arrays estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 have been confirmed for the first time in clinical Proteus isolates. Beta-lactamase genes and PMQR were investigated, and bla TEM-1 and bla OXA-1 were the most common, with qnrD and aac (6')-Ib-cr also being dominant.

Genome-wide analysis of the potato Hsp20 gene family: identification, genomic organization and expression profiles in response to heat stress.

BACKGROUND: Heat shock proteins (Hsps) are essential components in plant tolerance mechanism under various abiotic stresses. Hsp20 is the major family of heat shock proteins, but little of Hsp20 family is known in potato (Solanum tuberosum), which is an important vegetable crop that is thermosensitive. RESULTS: To reveal the mechanisms of potato Hsp20s coping with abiotic stresses, analyses of the potato Hsp20 gene family were conducted using bioinformatics-based methods. In total, 48 putative potato Hsp20 genes (StHsp20s) were identified and named according to their chromosomal locations. A sequence analysis revealed that most StHsp20 genes (89.6%) possessed no, or only one, intron. A phylogenetic analysis indicated that all of the StHsp20 genes, except 10, were grouped into 12 subfamilies. The 48 StHsp20 genes were randomly distributed on 12 chromosomes. Nineteen tandem duplicated StHsp20s and one pair of segmental duplicated genes (StHsp20-15 and StHsp20-48) were identified. A cis-element analysis inferred that StHsp20s, except for StHsp20-41, possessed at least one stress response cis-element. A heatmap of the StHsp20 gene family showed that the genes, except for StHsp20-2 and StHsp20-45, were expressed in various tissues and organs. Real-time quantitative PCR was used to detect the expression level of StHsp20 genes and demonstrated that the genes responded to multiple abiotic stresses, such as heat, salt or drought stress. The relative expression levels of 14 StHsp20 genes (StHsp20-4, 6, 7, 9, 20, 21, 33, 34, 35, 37, 41, 43, 44 and 46) were significantly up-regulated (more than 100-fold) under heat stress. CONCLUSIONS: These results provide valuable information for clarifying the evolutionary relationship of the StHsp20 family and in aiding functional characterization of StHsp20 genes in further research.

Genome-Wide Identification and Characterization of the Potato bHLH Transcription Factor Family.

Plant basic/helix-loop-helix (bHLH) transcription factors participate in a number of biological processes, such as growth, development and abiotic stress responses. The bHLH family has been identified in many plants, and several bHLH transcription factors have been functionally characterized in Arabidopsis. However, no systematic identification of bHLH family members has been reported in potato (Solanum tuberosum). Here, 124 StbHLH genes were identified and named according to their chromosomal locations. The intron numbers varied from zero to seven. Most StbHLH proteins had the highly conserved intron phase 0, which accounted for 86.2% of the introns. According to the Neighbor-joining phylogenetic tree, 259 bHLH proteins acquired from Arabidopsis and potato were divided into 15 groups. All of the StbHLH genes were randomly distributed on 12 chromosomes, and 20 tandem duplicated genes and four pairs of duplicated gene segments were detected in the StbHLH family. The gene ontology (GO) analysis revealed that StbHLH mainly function in protein and DNA binding. Through the RNA-seq and quantitative real time PCR (qRT-PCR) analyses, StbHLH were found to be expressed in various tissues and to respond to abiotic stresses, including salt, drought and heat. StbHLH1, 41 and 60 were highly expressed in flower tissues, and were predicted to be involved in flower development by GO annotation. StbHLH45 was highly expressed in salt, drought and heat stress, which suggested its important role in abiotic stress response. The results provide comprehensive information for further analyses of the molecular functions of the StbHLH gene family.

Genome-wide identification of the potato WRKY transcription factor family.

WRKY transcription factors play pivotal roles in regulation of stress responses. This study identified 79 WRKY genes in potato (Solanum tuberosum). Based on multiple sequence alignment and phylogenetic relationships, WRKY genes were classified into three major groups. The majority of WRKY genes belonged to Group II (52 StWRKYs), Group III had 14 and Group I consisted of 13. The phylogenetic tree further classified Group II into five sub-groups. All StWRKY genes except StWRKY79 were mapped on potato chromosomes, with eight tandem duplication gene pairs and seven segmental duplication gene pairs found from StWRKY family genes. The expression analysis of 22 StWRKYs showed their differential expression levels under various stress conditions. Cis-element prediction showed that a large number of elements related to drought, heat and salicylic acid were present in the promotor regions of StWRKY genes. The expression analysis indicated that seven StWRKYs seemed to respond to stress (heat, drought and salinity) and salicylic acid treatment. These genes are candidates for abiotic stress signaling for further research.

Bacterial magnetic particles improve testes-mediated transgene efficiency in mice.

Nano-scaled materials have been proved to be ideal DNA carriers for transgene. Bacterial magnetic particles (BMPs) help to reduce the toxicity of polyethylenimine (PEI), an efficient gene-transferring agent, and assist tissue transgene ex vivo. Here, the effectiveness of the BMP-PEI complex-conjugated foreign DNAs (BPDs) in promoting testes-mediated gene transfer (TMGT) in mouse was compared with that of liposome-conjugated foreign DNAs. The results proved that through testes injection, the clusters of BPDs successfully reached the cytoplasm and the nuclear of spermatogenesis cell, and expressed in testes of transgene founder mice. Additionally, the ratio of founder mice obtained from BPDs (88%) is about 3 times higher than the control (25%) (p < 0.05). Interestingly, the motility of sperms recovered from epididymis of the founder mice from BPD group were significantly improved, as compared with the control (p < 0.01). Based on classic breeding, the ratio of transgene mice within the first filial was significantly higher in BPDs compared with the control (73.8% versus 11.6%, p < 0.05). TMGT in this study did not produce visible histological changes in the testis. In conclusion, nano-scaled BPDs could be an alternative strategy for efficiently producing transgene mice in vivo.

Natriuretic peptide type C induces sperm attraction for fertilization in mouse.

Mammalian spermatozoa undergo selective movement along the isthmus of the oviduct to the ampulla during ovulation, which is a prerequisite for fertilization. The factor(s) that involves in selective spermatozoa movement is still unknown. In this study, we found that the oviductal epithelium in mouse ampulla expressed high levels of natriuretic peptide type C (NPPC) in the presence of ovulated oocyte-cumulus complexes (OCCs). Spermatozoa expressed NPPC receptor natriuretic peptide receptor 2 (NPR2, a guanylyl cyclase) on the midpiece of flagellum. NPPC increased intracellular levels of cGMP and Ca2+ of spermatozoa, and induced sperm accumulation in the capillary by attraction. Importantly, spermatozoa from Npr2 mutant mice were not attracted by NPPC, preventing fertilization in vivo. Oocyte-derived paracrine factors promoted the expression of Nppc mRNA in the ampulla. Therefore, NPPC secreted by oviductal ampulla attracts spermatozoa towards oocytes, which is essential for fertilization.

Epidermal Growth Factor-Mobilized Intracellular Calcium of Cumulus Cells Decreases Natriuretic Peptide Receptor 2 Affinity for Natriuretic Peptide Type C and Induces Oocyte Meiotic Resumption in the Mouse.

Natriuretic peptide type C (NPPC) activation of the guanylyl cyclase-linked natriuretic peptide receptor (NPR) 2 maintains oocyte meiotic arrest. Luteinizing hormone (LH)-dependent epidermal growth factor (EGF) receptor signaling elevates calcium of cumulus cells to inactivate NPR2, resulting in meiotic resumption. This study investigated the regulatory mechanism of calcium on NPR2 inactivation. In mouse ovarian follicles, LH, through the activation of EGF receptor, significantly elevated calcium levels in cumulus cells, but decreased the binding affinity of NPR2 for NPPC. In cultured cumulus-oocyte complexes, the activation of EGF receptor by EGF mobilized intracellular calcium of cumulus cells to decrease NPR2 affinity and cGMP levels, resulting in meiotic resumption. However, hormone treatments had not changed NPR2 protein levels. In addition, the removal of magnesium ions from the medium decreased the binding affinity of NPR2 for NPPC, resulting in a decrease in cGMP levels and meiotic resumption. It is concluded that magnesium ions are required to maintain functional NPR2, and that LH-dependent EGF receptor signaling mobilizes intracellular calcium of cumulus cells to reduce NPPC-NPR2 interaction that is required for meiotic resumption.