Impact of Huanglongbing Pathogen Infection on the Amino Acid Composition in Both Citrus Plants and the Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) Diaphorina citri is the main vector of the pathogen Candidatus Liberibacter asiaticus (CLas), which is the causal agent of citrus Huanglongbing disease. Feeding by both ACP nymphs and adults on host plants allows them to obtain nutrition. Therefore, the nutritional content within the plant phloem is of much importance for the development and reproduction of ACP. The infection by pathogenic microbiomes may affect the amino acid contents of their host plants and then indirectly affect the biology of sap-feeding insects. In this study, we investigated the amino acid contents and their proportions in both CLas-infected and CLas-free citrus plants, ACP adults, and also in honeydew produced by ACP nymphs. Results showed that infection by CLas had a large impact on the amino acid species and proportion in all the tested target plants, ACP adults, and in the honeydew of ACP nymphs. The content of total amino acids in CLas-infected citrus was much higher than that of CLas-free citrus. However, CLas infection significantly reduced the proportion of essential amino acids (EAAs) in these plants. When feeding on CLas-infected citrus plants, ACP adults absorbed less total amino acids than those adults feeding on healthy plants, but the proportion of EAAs was significantly higher when they fed on CLas-infected citrus plants. The proportion of EAAs also significantly increased in the honeydew secreted by ACP nymphs that fed on CLas-infected citrus plants. However, EAA detection in the honeydew of ACP nymphs indicated that the utilization rate of EAAs by CLas positive ACP nymphs was reduced. Our study has revealed that CLas infection significantly affects the contents, proportion, and utilization efficiency of different amino acids in citrus plants, ACP adults, and nymphs, leading to a developmental pattern of ACP that is more conducive to CLas transmission.

Parasitoid vectors a plant pathogen, potentially diminishing the benefits it confers as a biological control agent.

Huanglongbing (HLB) is a destructive disease of citrus primarily transmitted by the Asian citrus psyllid (ACP). Biocontrol of ACP is an environmentally sustainable alternative to chemicals. However, the risk of parasitoid rational application in ACP biocontrol has never been evaluated. Here we show, the dominant parasitoid of ACP, Tamarixia radiata, can acquire the HLB pathogen Candidatus Liberibacter asiaticus (CLas) and transmit it horizontally when probing ACP nymphs. If these ACP nymphs survive the probing, develop to adults and move to healthy plants, CLas can be transmitted to citrus leaves during feeding. We illustrate the formerly unrecognized risk that a parasitoid can potentially serve as a phoretic vector of the pathogen transmitted by its host, thus potentially diminishing some of the benefits it confers via biocontrol. Our findings present a significant caution to the strategy of using parasitoids in orchards with different infection status of insect-vectored pathogens.

Dimensions of the optic chiasm: quantitative ultrasound comparison between fetuses with anophthalmia/microphthalmia and normal fetuses.

BACKGROUND: The precise pathogenesis of anophthalmia/microphthalmia remains unknown. Prenatal observation of the optic chiasm in fetuses with this malformation would assist in understanding the embryonic development of the condition. The present study aimed to establish the normal fetal size ranges of decussation of the optic chiasm, optic nerves, and optic tracts in the axial plane using two-dimensional transabdominal ultrasound throughout gestation and to compare these ranges to the corresponding values in fetuses with anophthalmia/microphthalmia. METHODS: In total, 310 normal fetuses and 16 fetuses with anophthalmia/microphthalmia were included in this study. The widths of the decussation of the optic chiasm, optic nerves, and optic tracts of normal fetuses at 19-40 weeks' gestation were measured in the axial plane by two-dimensional transabdominal ultrasound. The same widths were retrospectively measured in the axial plane using three-dimensional ultrasound in fetuses with anophthalmia/microphthalmia and compared to the results from the normal fetuses. RESULTS: The decussation, optic nerves, and optic tracts of 310 normal fetuses were measured. The normal widths of the decussation of the optic chiasm, optic nerves, and optic tracts increased linearly with gestational age. The interobserver and intraobserver reproducibility was excellent for the decussation but relatively low for the optic nerves and optic tracts. The optic nerve width of fetuses with anophthalmia/microphthalmia was significantly smaller than that of normal fetuses (P<0.001), but the widths of the decussation (P=0.061) and optic tracts (P=0.053) were not significantly different between the two groups. CONCLUSIONS: The normal ranges of the decussation of the optic chiasm, optic nerves, and optic tracts established in this study can provide a quantitative basis for prenatal evaluation of the optic pathway. Fetal anophthalmia/microphthalmia may be associated with optic nerve hypoplasia.

Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri.

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiate and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm, ΔCt, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage; PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen); EF1A and ArgK between two populations; ß-tubulin and EF1A for different temperatures (5, 15, 25, 35 °C); and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

Quantitative elastography of cervical stiffness during the three trimesters of pregnancy with a semiautomatic measurement program: A longitudinal prospective pilot study.

AIM: To assess the reproducibility of a semiautomatic quantification tool for cervical stiffness and evaluate the normal changes in cervical elasticity during the three trimesters of pregnancy. METHODS: This longitudinal prospective pilot study evaluated cervical elasticity during the three trimesters of pregnancy (11-14, 20-24 and 28-32 weeks) in women with singleton pregnancies. Women with a history of conization, cerclage, cervical Naboth cysts (diameter > 10 mm), cervical tumors, or uterine malformation were excluded. A semiautomatic tool was used to evaluate the stiffness of the whole cervix and the internal and external cervical os with multiple quantitative elasticity parameters and the cervical length (CL) on the sagittal view via transvaginal elastography. Intraclass correlation coefficients (ICC) and Bland-Altman analysis were used to assess intra- and interobserver variability. E-Cervix parameters during the three trimesters were compared using the Friedman test. RESULTS: In total, 217 women with 651 strain examinations during the three trimesters were included. The intra- and interobserver ICC for the E-Cervix parameters ranged from 0.947 to 0.991 and 0.855 to 0.989, respectively. There were significant differences in all parameters among the three trimesters. Cervical elasticity showed significant softening and became heterogeneous during the three trimesters. The median CL was significantly shorter in the first trimester than in the second and third trimesters (P = 0.004, P < 0.001). CONCLUSION: E-Cervix provides a graphical tool for operators to easily define regions of interest and obtain multiple repeatable measures of elasticity. The normal references for E-Cervix parameters during the three trimesters reflect the physiological cervical changes during pregnancy.

Evaluation of Cervical Elastography for Prediction of Spontaneous Preterm Birth in Low-Risk Women: A Prospective Study.

OBJECTIVES: The aim of this study was to determine whether cervical elastographic parameters in addition to cervical length (CL) during the 3 trimesters of pregnancy would be predictive of spontaneous preterm birth (sPTB) among low-risk women. METHODS: This work was a prospective nested case-control study evaluating cervical elastographic parameters and CL in low-risk women during the 3 trimesters of pregnancy. A binary logistic regression analysis was used to calculate significant covariates for prediction of sPTB. The area under the curve of the prediction model was calculated by using a receiver operating characteristic curve. RESULTS: There were 286 women (26 cases and 260 controls) included in the analysis. The parameters of cervical elasticity became softened and heterogeneous during the 3 trimesters of pregnancy in both women with and without sPTB. The differences in the mean strain value at the internal os of the cervix (IOS), ratio (strain ratio of the internal os to the external os) during the second trimester and the IOS during the third trimester between the groups had statistical significance (P < .01; P = .01; P < .01, respectively). The CL had no association with sPTB during the 3 trimesters. The IOS during the second trimester was a better predictor of sPTB, with an area under the curve of 0.730, and sensitivity was 72.73%. CONCLUSIONS: We observed multiple elastographic parameters and demonstrated the physiologic changes in the cervix during the 3 trimesters of pregnancy. Furthermore, we found that the IOS during the second trimester can be helpful in predicting sPTB. However, the CL had no association with sPTB during the 3 trimesters of pregnancy.

Tamarixia radiata Behaviour is Influenced by Volatiles from Both Plants and Diaphorina citri Nymphs.

Tamarixia radiata (Waterston) is an important ectoparasitoid of the Asian citrus psyllid, Diaphorina citri Kuwayama, a globally destructive pest of citrus. In the present study, a Y-tube olfactometer was employed to investigate whether the parasitoid T. radiata is capable of utilizing the odour source emitted by both plants and insect hosts during its foraging. The odour sources included Murraya paniculata (L.) shoots, 1st, 2nd, 3rd, 4th, and 5th D. citri instar nymphs, both individually and in combinations. Moreover, nymph-stage choice for parasitism, including 3rd, 4th, and 5th D. citri instar nymphs, was carried out. The results indicated that female T. radiata were only significantly attracted to volatiles emitted by M. paniculata shoots, 3rd, 4th, and 5th instar nymphs of D. citri, but could not distinguish between them. T. radiata males were not attracted by odours sourced from any instar D. citri nymphs. Female T. radiata adults exhibited a significant preference to later instar nymphal stages of D. citri for oviposition. The results from this study can be used to guide further investigations on the searching behaviour of this parasitoid and its utilization in D. citri biocontrol.

A Cell-Permeant Mimetic of NMN Activates SARM1 to Produce Cyclic ADP-Ribose and Induce Non-apoptotic Cell Death.

SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca2+ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells.

Synthesis and Evaluation of Novel Neamine-Nucleoside Conjugates as Potential Antibiotic Targets for Escherichia coli 16S Ribosomal RNA.

Based on the nucleobase rich character of the binding pocket of A-site 16S ribosomal RNA of Escherichia coli, it was proposed that the neamine moiety of synthesized Neamine-nucleoside conjugates could bind to the groove of RNA while the nucleobase moiety would bind specifically to the sequence of the 16S rRNA A-site fragment. Thus the designed conjugate compound 5 was found to have the same dissociation constant as neamine for binding to 16S rRNA and the neamine-amino acid substituted nucleoside conjugate 8 and 9 showed 6.3 and 4.8 times greater RNA binding affinity, respectively, as compared with neamine. The results obtained successfully demonstrate the need for chemically modifying neamine and probe the changes induced using NMR protocols to assist in the discovery of new aminoglycoside antibiotics.

Identification of a new Cordyceps javanica fungus isolate and its toxicity evaluation against Asian citrus psyllid.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most serious pest of citrus worldwide. It acts as a vector for a group of phloem-limited bacteria (Candidatus Liberibacter spp.) that causes Huanglongbing (HLB) disease. Thus, D. citri management is an important strategy against HLB, and biological control is currently considered as the most effective method because of the unsustainable and negative side effects of chemical control. Here, we isolated a new strain of entomopathogenic fungus, Cordyceps javanica (GZQ-1), from one cadaver of D. citri adult based on its morphological and phylogenetic data. Five conidial concentrations of the C. javanica pathogen (1 × 103 , 1 × 104 , 1 × 105 , 1 × 106 , and 1 × 107 conidia/ml) were assessed against six life stages of D. citri (1st-5th instar nymphs and adults). Results showed that C. javanica GZQ-1 was highly pathogenic to D. citri nymphs (69.49%-90.87% mortality) and adults (69.98% mortality). The LC50 and LT50 values of C. javanica against 1st-2nd instar (younger), 3rd-4th instar (middle aged), 5th instar (older), and adults were 1.20 × 105 , 1.10 × 106 , 4.47 × 106 , 8.12 × 106 conidia/ml and 4.25, 4.51, 5.17, 5.49 days, respectively. Moreover, glasshouse experiments indicated that this C. javanica GZQ-1 caused higher infection rates of D. citri adults compared to two other fungal strains we previously isolated in the laboratory, Cordyceps fumosorosea (IF010) and Metarhizium anisopliae (CNGD7).

Chemoradiotherapy Versus Radiotherapy Alone in Stage II Nasopharyngeal Carcinoma: A Systemic Review and Meta-analysis of 2138 Patients.

Background: To explore the value of chemoradiotherapy (CRT) in stage II nasopharyngeal carcinoma (NPC) compared to radiotherapy (RT) alone which includes two-dimensional radiotherapy (2D-RT) and intensity-modulated radiotherapy (IMRT). Methods: All topic-related comparative articles were identified by a comprehensive search of public databases (MEDLINE, EMBASE, Cochrane Library and CBMdisc). The primary outcomes were overall survival (OS), loco-regional relapse-free survival (LRRFS) and distant metastasis-free survival (DMFS). Secondary outcomes were grade 3-4 acute toxicity events. We performed subgroup analysis of CRT versus 2D-RT/IMRT alone to investigate the optimal modality. Sensitivity analysis focused on CRT versus IMRT alone was used to assess stability of the study results. Results: Eleven comparative studies (2138 patients) were eligible. CRT had significantly higher OS (HR = 0.67, 95% CI = 0.45-0.98, P = 0.04) and LRRFS (HR = 0.61, 95% CI = 0.46-0.80, P = 0.0003) than RT alone, but no significant difference was observed in DMFS (HR = 0.83, 95% CI = 0.52-1.31, P = 0.41). Meanwhile, CRT was associated with higher frequencies of grade 3-4 leukopenia, mucositis and nausea (P = 0.005, 0.03, < 0.0001, respectively). Subgroup analysis showed that IMRT alone could achieve equivalent OS, LRRFS and DMFS compared to CRT (P = 0.14, 0.06, 0.89, respectively). Significant value was only observed in LRRFS for CRT compared to 2D-RT alone (P = 0.01). Sensitivity analysis for the comparison of CRT and IMRT alone demonstrated generally stable outcomes, in support of the final conclusions. Conclusions: In the treatment of patients with stage II NPC, CRT was better than 2D-RT alone with significant benefit in LRRFS. IMRT alone was superior to CRT with equivalent survival outcomes and fewer grade 3-4 acute toxicities.

Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence.

Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a "core" sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive "core" sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.

Identifying Glyceraldehyde 3-Phosphate Dehydrogenase as a Cyclic Adenosine Diphosphoribose Binding Protein by Photoaffinity Protein-Ligand Labeling Approach.

Cyclic adenosine diphosphoribose (cADPR), an endogenous nucleotide derived from nicotinamide adenine dinucleotide (NAD+), mobilizes Ca2+ release from endoplasmic reticulum (ER) via ryanodine receptors (RyRs), yet the bridging protein(s) between cADPR and RyRs remain(s) unknown. Here we synthesized a novel photoaffinity labeling (PAL) cADPR agonist, PAL-cIDPRE, and subsequently applied it to purify its binding proteins in human Jurkat T cells. We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of the cADPR binding protein(s), characterized the binding affinity between cADPR and GAPDH in vitro by surface plasmon resonance (SPR) assay, and mapped cADPR's binding sites in GAPDH. We further demonstrated that cADPR induces the transient interaction between GAPDH and RyRs in vivo and that GAPDH knockdown abolished cADPR-induced Ca2+ release. However, GAPDH did not catalyze cADPR into any other known or novel compound(s). In summary, our data clearly indicate that GAPDH is the long-sought-after cADPR binding protein and is required for cADPR-mediated Ca2+ mobilization from ER via RyRs.

The decreased N6-methyladenine DNA modification in cancer cells.

N6-methyladenine (6 mA) is a recently characterized DNA modification in mammalian genomes, although its biological importance remains to be resolved. Using a highly sensitive HPLC/MS/MS approach, here we report regulation of 6 mA modification in mammalian cells. To these aspects, down-regulation of 6 mA modification was first characterized in human cancer cells and tissues, relative to their normal controls. In contrast to the relative stable 5 mC modification, a dramatic decrease of 6 mA modification was found, showing that 6 mA is the most regulated DNA modification in cancers. In addition to the regulation in cancer cells, a hundreds-fold increase of 6 mA modification was found for in vitro cultured human cells, relative to the in vivo cells. This up-regulation was also confirmed with in vitro cultured mouse cells. Taken together, our study revealed distinct 6 mA modification profiles in the cancer and cultured cells. Considering its distinct regulation from that of 5 mC, our study suggests that 6 mA DNA modification may play a crucial role in cell fate transition of mammalian cells.

tRNA modification profiles of the fast-proliferating cancer cells.

Despite the recent progress in RNA modification study, a comprehensive modification profile is still lacking for mammalian cells. Using a quantitative HPLC/MS/MS assay, we present here a study where RNA modifications are examined in term of the major RNA species. With paired slow- and fast-proliferating cell lines, distinct RNA modification profiles are first revealed for diverse RNA species. Compared to mRNAs, increased ribose and nucleobase modifications are shown for the highly-structured tRNAs and rRNAs, lending support to their contribution to the formation of high-order structures. This study also reveals a dynamic tRNA modification profile in the fast-proliferating cells. In addition to cultured cells, this unique tRNA profile has been further confirmed with endometrial cancers and their adjacent normal tissues. Taken together, the results indicate that tRNA is a actively regulated RNA species in the fast-proliferating cancer cells, and suggest that they may play a more active role in biological process than expected.

Inter-molecular ß-sheet structure facilitates lung-targeting siRNA delivery.

Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-ß, which was able to form inter-molecular ß-sheet structures. Results showed that K-ß peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.

[Treatment of triple negative breast cancer cells with combination of mTOR1/2 inhibitor AZD8055 and MEK1/2 inhibitor PD0325901].

MEK inhibition activates PI3K/AKT/mTOR pathway in triple negative breast cancer (TNBC) cell lines. Combination of PI3K inhibitor and MEK1/2 inhibitor is not appropriate for PI3K inhibitor insensitive TNBC cell lines. This study was designed to investigate the effects of dual treatments with mTOR1/2 inhibitor AZD8055 and MEK1/2 inhibitor PD0325901 in MDA-MB-435 cell line. MEK1/2 inhibition led to activation of AKT, which is the downstream signaling protein of PI3K pathway. The combination inhibited the phosphorylation of AKT and therefore abolished the feedback interaction of two pathways. Cell proliferation assay and DNA replication assay demonstrated that the dual treatments led to a significant synergistic inhibition of cell cycle progression and cell proliferation.

[The synthesis of purine derivatives and its inhibitory activity on CD38 NADase].

CD38 is a multifunctional enzyme expressed in a variety of mammalian tissues, its catalytic activity was involved in a wide range of physiological processes. Based on the reported inhibitor of human CD38 NADase, 33 purine derivatives were designed and synthesized. The biological activity assay showed that compounds 20 and 38 exhibited almost the same extent of inhibitory activities on human CD38 NADase as the lead compound H2. The results also revealed that small substituents at C-6 of purine ring gave no obvious effect on inhibitory activity, but phenylpropionyl moiety at N-2 could affect the binding mode of the compound with CD38. This study provides a reliable basis for future rational design of inhibitors for CD38.

CD38 Is Required for Neural Differentiation of Mouse Embryonic Stem Cells by Modulating Reactive Oxygen Species.

CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca(2+) mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production.

[Design and synthesis of photoaffinity biotin labelled 2'-O-propargyl-guanosine].

Photoaffinity labeling is widely applied to demonstrate targets of small molecule ligands. In this paper, biotin photoaffinity labeled molecule with propargyl group 1 has been designed and synthesized, followed it's labeling of N2-acetyl-2'-O-propargyl guanosine 9 by "click chemistry". This technology presents delight development potential in labeling of second messenger cyclic nucleotide, antisense oligonucleotide or siRNA.