CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 is an RNA-binding protein controlling plant immunity via an RNA processing complex.

Plant innate immunity is capable of combating diverse and ever evolving pathogens. The plasticity of innate immunity could be boosted by RNA processing. Arabidopsis thaliana CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5), a key negative immune regulator, is a component of the nuclear pore complex. Here we further identified CPR5 as a component of RNA processing complexes. Through genetic screening, we found that RNA splicing activator NineTeen Complex and RNA polyadenylation factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR, coordinately function downstream of CPR5 to activate plant immunity. CPR5 and these two regulators form a complex that is localized in nuclear speckles, an RNA processing organelle. Intriguingly, we found that CPR5 is an RNA-binding protein belonging to the Transformer 2 (Tra2) subfamily of the serine/arginine-rich family. The RNA recognition motif of CPR5 protein binds the Tra2-targeted RNA sequence in vitro and is functionally replaceable by those of Tra2 subfamily proteins. In planta, it binds RNAs of CPR5-regulated alternatively spliced genes (ASGs) identified by RNA-seq. ARGONAUTE 1 (AGO1) is one of the ASGs and, consistent with this, the ago1 mutant suppresses the cpr5 phenotype. These findings reveal that CPR5 is an RNA-binding protein linking RNA processing with plant immunity.

Sapap4 deficiency leads to postsynaptic defects and abnormal behaviors relevant to hyperkinetic neuropsychiatric disorder in mice.

Postsynaptic proteins play critical roles in synaptic development, function, and plasticity. Dysfunction of postsynaptic proteins is strongly linked to neurodevelopmental and psychiatric disorders. SAP90/PSD95-associated protein 4 (SAPAP4; also known as DLGAP4) is a key component of the PSD95-SAPAP-SHANK excitatory postsynaptic scaffolding complex, which plays important roles at synapses. However, the exact function of the SAPAP4 protein in the brain is poorly understood. Here, we report that Sapap4 knockout (KO) mice have reduced spine density in the prefrontal cortex and abnormal compositions of key postsynaptic proteins in the postsynaptic density (PSD) including reduced PSD95, GluR1, and GluR2 as well as increased SHANK3. These synaptic defects are accompanied by a cluster of abnormal behaviors including hyperactivity, impulsivity, reduced despair/depression-like behavior, hypersensitivity to low dose of amphetamine, memory deficits, and decreased prepulse inhibition, which are reminiscent of mania. Furthermore, the hyperactivity of Sapap4 KO mice could be partially rescued by valproate, a mood stabilizer used for mania treatment in humans. Together, our findings provide evidence that SAPAP4 plays an important role at synapses and reinforce the view that dysfunction of the postsynaptic scaffolding protein SAPAP4 may contribute to the pathogenesis of hyperkinetic neuropsychiatric disorder.

Effects of kisspeptin on pathogenesis and energy metabolism in polycystic ovarian syndrome (PCOS).

Kisspeptin has been shown to participate in the regulation of pituitary hormone secretion and energy metabolism. In PCOS patients, there are disorders in pituitary hormone secretion and energy metabolism. The aim of this study was to investigate the serum kisspeptin and its relationship with abnormal metabolism in PCOS. This restrospective case-control study included 73 cases with PCOS and 63 control cases. All subjects were divided into obese and nonobese groups based on BMI. The serum kisspeptin levels, Cor, DHEA-S, plasma concentrations of glucose were tested. We found that the level of kisspeptin in PCOS group was higher than it in control group. The kisspeptin levels in nonobese PCOS group increased most obviously over than the other groups. The kisspeptin levels of all the subjects were positively correlated with LH levels, negatively correlated with the glucose-AUC, the insulin-AUC, and triglyceride levels. The findings of this study suggest that kisspeptin may play an important role in ovulation disorders in PCOS patients through regulating the level of LH and it could regulate the body's energy metabolism by regulating glucose and lipid metabolism.

Effect of Ostomy Care Team Intervention on Patients With Ileal Conduit.

PURPOSE: The purpose of this study was to investigate the effect of a nurse-led multicomponent intervention on ostomy-related complications, self-efficacy, and health-related quality of life in patients with an ileal conduit. DESIGN: Randomized controlled trial. SAMPLE AND SETTING: Forty-six patients who underwent radical cystectomy and creation of an ileal conduit participated in the trial; data were collected over a 6-month period. The study setting was Shanghai Pudong Hospital, affiliated with Fudan University, located in Shanghai, China. METHODS: Participants were randomly allocated to an experimental or control group. Participants in the control group received routine care over a 6-month period following ostomy surgery, while those in the experimental group received a nurse-led, multicomponent, structured intervention delivered by an ostomy care team. The Chinese language versions of the Stoma Self-Efficacy Scale (SSES) and the City of Hope Quality of Life-Ostomy (COHQOL-O) questionnaire were used to assess self-efficacy in stoma care and health-related quality of life. Ostomy-related complications including peristomal moisture-associated skin damage and uric acid crystal deposits in the peristomal area were also assessed. Fisher's exact test was used to compare the incidence of ostomy-related complications between the 2 groups. Independent-samples t tests were used to compare SSES and COHQOL-O scores. RESULTS: No statistically significant differences were found between demographic characteristics of the control and experimental groups. After 6 months, the incidence of complications was significantly lower in the experimental group as compared to the control group (4.35% vs 30.43%, P = .047). In addition, the mean SSES score was significantly higher in the experimental group (indicating greater self-efficacy in stoma care) (107.13 ± 11.87 vs 85.65 ± 12.87, P = .000), and the mean COHQOL-O score was also significantly higher in the experimental group, indicating higher health-related quality of life (154.48 ± 16.01 vs 138.26 ± 13.42, P = .001). CONCLUSION: The nurse-led multicomponent intervention provided by the ostomy care team reduced ostomy-related complications and improved the self-efficacy level and health-related quality of life in persons with a new urostomy.

Statistical considerations regarding correlated lots in analytical biosimilar equivalence test.

In the evaluation of the analytical similarity data, an equivalence testing approach for most critical and quantitative quality attributes, which are assigned to Tier 1 in their proposed three-tier approach, was proposed. The Food and Drug Administration (FDA) has recommended the proposed equivalence testing approach to sponsors through meeting comments for Pre-Investigational New Drug Applications (PINDs) and Investigational New Drug Applications (INDs) since 2014. The FDA has received some feedback on the statistical issues of potentially correlated reference lot values subjected to equivalence testing since independent and identical observations (lot values) from the proposed biosimilar product and the reference product are assumed. In this article, we describe one method for correcting the estimation bias of the reference variability so as to increase the equivalence margin and its modified versions for increasing the equivalence margin and correcting the standard errors in the confidence intervals, assuming that the lot values are correlated under a few known correlation matrices. Our comparisons between these correcting methods and no correction for bias in the reference variability under several assumed correlation structures indicate that all correcting methods would increase the type I error rate dramatically but only improve the power slightly for most of the simulated scenarios. For some particular simulated cases, the type I error rate can be extremely large (e.g., 59%) if the guessed correlation is larger than the assumed correlation. Since the source of a reference drug product lot is unknown in nature, correlation between lots is a design issue. Hence, to obtain independent reference lot values by purchasing the reference lots at a wide time window often is a design remedy for correlated reference lot values.

Exact test-based approach for equivalence test with parameter margin.

The equivalence test has a wide range of applications in pharmaceutical statistics which we need to test for the similarity between two groups. In recent years, the equivalence test has been used in assessing the analytical similarity between a proposed biosimilar product and a reference product. More specifically, the mean values of the two products for a given quality attribute are compared against an equivalence margin in the form of ±f × σR, where ± f × σ R is a function of the reference variability. In practice, this margin is unknown and is estimated from the sample as ±f × SR. If we use this estimated margin with the classic t-test statistic on the equivalence test for the means, both Type I and Type II error rates may inflate. To resolve this issue, we develop an exact-based test method and compare this method with other proposed methods, such as the Wald test, the constrained Wald test, and the Generalized Pivotal Quantity (GPQ) in terms of Type I error rate and power. Application of those methods on data analysis is also provided in this paper. This work focuses on the development and discussion of the general statistical methodology and is not limited to the application of analytical similarity.

Environmental Affection-Driven English Tense Analysis: A Healthcare Exercise-Based Corpus Case Study over Public English Environment.

Most international academic papers are written in English, and the use of tenses in English academic papers often follows some conventional rules. Automatically extracting and analyzing English tenses in scientific papers have begun to attract researchers' attention for the global environment. In the analysis of the English tense of scientific papers, consider that the neural network model that combines attention mechanism and sequential input network such as Long Short-Term Memory (LSTM) network has a long training time, low extraction accuracy, and cannot parallelize text input. We propose an environmental affection-driven English tense analysis model, which includes an attention mechanism and LSTM model and conducts a temporal analysis of English texts based on an affective computing model. In this paper, our proposed method is verified based on the self-built healthcare exercise-based corpus over public English environment. By comparison, the experimental results show that the method proposed in this paper has better performance than ordinary Convolutional Neural Network (CNN), Support Vector Machine (SVM), and LSTM based on attention mechanism.

Mental Health Management of English Teachers in English Teaching Under the COVID-19 Era.

Background: The COVID-19 pandemic has brought new challenges and attention to the mental health of all social groups, making mental health increasingly necessary and important. However, people only focus on the mental health of undergraduates, and the mental health of teachers has not received much attention from society. College teachers are the backbone of the teachers' group, and their mental health not only affects the teaching quality and research level but also plays an important role in the mental health and personality development of undergraduates. Method: During the COVID-19 pandemic, online teaching is a major challenge for college teachers, especially English teachers. To this end, this article proposes a bipartite graph convolutional network (BGCN) model based on the psychological test questionnaire and its structural characteristics for the recognition of the mental health crisis. Results: Experimental results show that the proposed BGCN model is superior to neural network algorithms and other machine learning algorithms in accuracy, precision, F1, and recall and can be well used for the mental health management of English teachers in the era of COVID-19.

A small-molecule oxocarbazate inhibitor of human cathepsin L blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293T cells.

A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of severe acute respiratory syndrome (SARS) coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC(50) from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single-step competitive inhibition model provided rate constants of k(on) = 153,000 M(-1)s(-1) and k(off) = 4.40 x 10(-5) s(-1) (K(i) = 0.29 nM). The compound also displayed cathepsin L/B selectivity of >700-fold and was nontoxic to human aortic endothelial cells at 100 muM. The oxocarbazate and a related thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC(50) = 273 +/- 49 nM) and Ebola virus (IC(50) = 193 +/- 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (DCG-04) was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor-treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.

A functional analysis of N-glycosylation-related genes on sialylation of recombinant erythropoietin in six commonly used mammalian cell lines.

Significant efforts have been made to improve the sialylation of recombinant glycoproteins with the aim of extending their in vivo circulation time. Here, we report a systematic functional analysis of 31 N-glycosylation-related genes on sialylation of recombinant EPO in six cell lines. BHK and CHO cells were found to sialylate recombinant EPO most effectively. None of the 31 genes, individually or in combination, was able to improve EPO sialylation in these cells. HEK293, Cos-7 and 3T3 cells showed intermediate sialylation capabilities, whereas NS0 cells sialylated recombinant EPO poorly. Overexpression of ST6GalI, ST3GalIII or ST3GalIV, but not ST3GalVI, was able to improve EPO sialylation in these four cell lines. qRT-PCR experiments revealed that ST3GalIII and ST3GalIV are indeed under expressed in HEK293, 3T3 and NS0 cells. Co-expression of upstream glycogenes failed to synergize with these sialyltransferases to further enhance sialylation, suggesting that the upstream glycogenes are all expressed at sufficient levels.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants.

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.

Molecular Dynamics Simulation on Mechanical and Piezoelectric Properties of Boron Nitride Honeycomb Structures.

Boron nitride honeycomb structure is a new three-dimensional material similar to carbon honeycomb, which has attracted a great deal of attention due to its special structure and properties. In this paper, the tensile mechanical properties of boron nitride honeycomb structures in the zigzag, armchair and axial directions are studied at room temperature by using molecular dynamics simulations. Effects of temperature and strain rate on mechanical properties are also discussed. According to the observed tensile mechanical properties, the piezoelectric effect in the zigzag direction was analyzed for boron nitride honeycomb structures. The obtained results showed that the failure strains of boron nitride honeycomb structures under tensile loading were up to 0.83, 0.78 and 0.55 in the armchair, zigzag and axial directions, respectively, at room temperature. These findings indicated that boron nitride honeycomb structures have excellent ductility at room temperature. Moreover, temperature had a significant effect on the mechanical and tensile mechanical properties of boron nitride honeycomb structures, which can be improved by lowering the temperature within a certain range. In addition, strain rate affected the maximum tensile strength and failure strain of boron nitride honeycomb structures. Furthermore, due to the unique polarization of boron nitride honeycomb structures, they possessed an excellent piezoelectric effect. The piezoelectric coefficient e obtained from molecular dynamics was 0.702   C / m 2 , which was lower than that of the monolayer boron nitride honeycomb structures, e = 0.79   C / m 2 . Such excellent piezoelectric properties and failure strain detected in boron nitride honeycomb structures suggest a broad prospect for the application of these new materials in novel nanodevices with ultrahigh tensile mechanical properties and ultralight-weight materials.

The Canonical E2Fs Are Required for Germline Development in Arabidopsis.

A number of cell fate determinations, including cell division, cell differentiation, and programmed cell death, intensely occur during plant germline development. How these cell fate determinations are regulated remains largely unclear. The transcription factor E2F is a core cell cycle regulator. Here we show that the Arabidopsis canonical E2Fs, including E2Fa, E2Fb, and E2Fc, play a redundant role in plant germline development. The e2fa e2fb e2fc (e2fabc) triple mutant is sterile, although its vegetative development appears normal. On the one hand, the e2fabc microspores undergo cell death during pollen mitosis. Microspores start to die at the bicellular stage. By the tricellular stage, the majority of the e2fabc microspores are degenerated. On the other hand, a wild type ovule often has one megaspore mother cell (MMC), whereas the majority of e2fabc ovules have two to three MMCs. The subsequent female gametogenesis of e2fabc mutant is aborted and the vacuole is severely impaired in the embryo sac. Analysis of transmission efficiency showed that the canonical E2Fs from both male and female gametophyte are essential for plant gametogenesis. Our study reveals that the canonical E2Fs are required for plant germline development, especially the pollen mitosis and the archesporial cell (AC)-MMC transition.

[Changes of the microcirculatory blood perfusion at "Feishu" (BL 13) in the COPD rats].

OBJECTIVE: To observe the change of the specificity of the microcirculatory blood perfusion at the area of "Feishu" (BL 13) in the rats of chronic obstructive pulmonary disease (COPD). METHODS: According to the random number table, 60 Wistar rats were divided into a 29 d model No. 1 group (C1 group), a 29 d normal control No.1 group (N1 group), a 89 d model No.2 group (C2 group) and a 89 d normal control No. 2 group (N2 group), 15 rats in each one. In the C1 and C2 groups, the smoking and intratracheal drops of endotoxin were used in combination to prepare COPD model. The rats were fed normally in the N1 and N2 groups. "Feishu" (BL 13), "Xinshu" (BL 15), the lateral site of "Feishu" (BL 13) and the lateral site of "Xinshu" (BL 15) were selected as the monitoring points. The pericam perfusion speckle imager (PeriCam PSI System) was adopted to monitor the microcirculatory perfusion unit (PU) at the monitoring points before and in 29 d and 89 d after modeling separately. RESULTS: Before modeling, the differences in PU were not significant at each monitoring point in comparison among the 4 groups and the differences were not significant among "Feishu" (BL 13) and "Xinshu" (BL 15) as well as their lateral sites (all P>0.05). After modeling, PU was increased at each monitoring point in the C1 and C2 groups (all P<0.05). PU in the C1 group was higher than the N1 group and that in the C2 group was lower than the N2 group, PU at each monitoring point in the C1 group were higher than the C2 group, indicating the significant differences (all P<0.05). In the C1 and C2 groups, the specific change occurred, in which PU at "Feishu" (BL 13) was higher than its lateral site. But such specific change did not happen in the N1 and N2 groups. CONCLUSION: PU at "Feishu" (BL 13) presents the specific change relevant with the sickness duration in the COPD rats.

LC/MS-based Intact IgG and Released Glycan Analysis for Bioprocessing Applications.

Robust plate based antibody glycan analysis platforms are urgently needed for biopharmaceutical development and manufacturing as well as for clinical biomarker research. A 96-well plate based workflow has been developed to analyze both intact IgG antibodies and released N-glycans using an Orbitrap Fusion Mass Spectrometer and an LC/MS method on the Waters UNIFI platform. Here, such a workflow including protein A purification, PNGaseF digestion, 2-AB labeling, and SPE clean-up is described. The measured IgG glycan profile is consistent with that obtained from non-plate based method and commercial kit and has the advantage of less hands-on time. Also the application of the workflow in cell culture monitoring and clonal selection work is demonstrated. Apart from checking the major glycan structure changes among clones, post translational modifications (PTMs) such as C-terminal lysine residue clipping and N-terminal pyroglutamic acid formation can also be deduced from the workflow.

Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells.

Influence of Human p53 on Plant Development.

Mammalian p53 is a super tumor suppressor and plays a key role in guarding genome from DNA damage. However, p53 has not been found in plants which do not bear cancer although they constantly expose to ionizing radiation of ultraviolet light. Here we introduced p53 into the model plant Arabidopsis and examined p53-conferred phenotype in plant. Most strikingly, p53 caused early senescence and fasciation. In plants, fasciation has been shown as a result of the elevated homologous DNA recombination. Consistently, a reporter with overlapping segments of the GUS gene (1445) showed that the frequency of homologous recombination was highly induced in p53-transgenic plants. In contrast to p53, SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), as a negative regulator of homologous recombination in plants, is not present in mammals. Comet assay and clonogenic survival assay demonstrated that SNI1 inhibited DNA damage repair caused by either ionizing radiation or hydroxyurea in human osteosarcoma U2OS cancer cells. RAD51D is a recombinase in homologous recombination and functions downstream of SNI1 in plants. Interestingly, p53 rendered the sni1 mutants madly branching of inflorescence, a phenotype of fasciation, whereas rad51d mutant fully suppressed the p53-induced phenotype, indicating that human p53 action in plant is mediated by the SNI1-RAD51D signaling pathway. The reciprocal species-swap tests of p53 and SNI1 in human and Arabidopsis manifest that these species-specific proteins play a common role in homologous recombination across kingdoms of animals and plants.

Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs.

Glycosylation of therapeutic proteins has a profound impact on their safety and efficacy. Many factors shape the glycosylation of biotherapeutics, ranging from expression systems and cell culture processes to downstream purification strategies. Various analytical technologies have been developed to address questions concerning different aspects of glycosylation. Informatics tools are also crucial for a systematic understanding of the glycosylation processes. Hence, an integrated approach is required to harness glycosylation for the production of optimal and consistent glycoprotein-based therapeutic drugs. Here, we review the latest developments and challenges in glycosylation analysis and control in the context of bioprocessing monoclonal antibodies.

IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains.

Aggregates in protein therapeutics like IgG monoclonal antibodies (mAb) are detrimental to product safety and efficacy. It has been reported that aggregates form in Chinese hamster ovary (CHO) cell lines expressing greater amount of heavy chain (HC) than light chain (LC). In this study, we observed that aggregates could form within the cells with excess HC and were partially secreted into the supernatant. The aggregates in the supernatant consisted of mainly HC and were partially dissociated under either reducing or denaturing conditions. Mutation of a predicted free cysteine on HC to prevent disulfide bonding did not reduce aggregation. Re-transfecting CHO cells with excess HC with more BiP, an important IgG molecular chaperone, partially reduced unwanted aggregates and fragments possibly by helping retain more incomplete products within the cell for either proper assembly or degradation. A second transfection of LC into CHO cells with excess HC to increase the LC expression to a level greater than the HC expression successfully removed all aggregates and fragments. mAb product aggregation in CHO cells with excess HC occur due to a combination of limited chaperones and LC:HC ratio. These results provide added insights to aggregate formation and would be useful for development of mAb cell lines with reduced aggregates.