Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

Quantitative Characterization of Protein N-Linked Core-Fucosylation by an Efficient Glycan Truncation Strategy.

Dysregulation of protein core-fucosylation plays a pivotal role in the onset, progression, and immunosuppression of cancer. However, analyzing core-fucosylation, especially the accurate determination of the core-fucosylation (CF) site occupancy ratio, remains challenging. To address these problems, we developed a truncation strategy that efficiently converts intact glycopeptides with hundreds of different glycans into two truncated forms, i.e., a monosaccharide HexNAc and a disaccharide HexNAc+core-fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of the CF occupancy ratio with high reproducibility. Notably, three times CF sites were identified using this strategy compared to conventional methods based on intact glycopeptides. Application of this platform to characterize protein core-fucosylation in two breast cancer cell lines, i.e., MDA-MB-231 and MCF7, yields a total of 1615 unique glycosites and about 900 CF sites from one single LC-MS/MS analysis. Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes and provides insights into developing new therapeutic strategies to aid precision medicine. Given the robust performance of this platform, it would have broad application in discovering novel biomarkers based on the CF glycosylation pattern, investigating cancer mechanisms, as well as detecting new intervention targets.

Novel players in organogenesis and flavonoid biosynthesis in cucumber glandular trichomes.

Glandular trichomes (GTs) are outgrowths of plant epidermal cells that secrete and store specialized secondary metabolites that protect plants against biotic and abiotic stresses and have economic importance for human use. While extensive work has been done to understand the molecular mechanisms of trichome organogenesis in Arabidopsis (Arabidopsis thaliana), which forms unicellular, nonglandular trichomes (NGTs), little is known about the mechanisms of GT development or regulation of secondary metabolites in plants with multicellular GTs. Here, we identified and functionally characterized genes associated with GT organogenesis and secondary metabolism in GTs of cucumber (Cucumis sativus). We developed a method for effective separation and isolation of cucumber GTs and NGTs. Transcriptomic and metabolomic analyses showed that flavonoid accumulation in cucumber GTs is positively associated with increased expression of related biosynthesis genes. We identified 67 GT development-related genes, the functions of 7 of which were validated by virus-induced gene silencing. We further validated the role of cucumber ECERIFERUM1 (CsCER1) in GT organogenesis by overexpression and RNA interference transgenic approaches. We further show that the transcription factor TINY BRANCHED HAIR (CsTBH) serves as a central regulator of flavonoid biosynthesis in cucumber GTs. Work from this study provides insight into the development of secondary metabolite biosynthesis in multicellular GTs.

GP-Marker facilitates the analysis of intact glycopeptide quantitative data at different levels.

Protein glycosylation is a highly heterogeneous post-translational modification that has been demonstrated to exhibit significant variations in various diseases. Due to the differential patterns observed in disease and healthy populations, the glycosylated proteins hold promise as early indicators for multiple diseases. With the continuous development of liquid chromatography-mass spectrometry (LC-MS) technology and spectrum analysis software, the sensitivity for the decipher of the tandem mass spectra of the glycopeptides carrying intact glycans, i.e., intact glycopeptides, enzymatic hydrolyzed from glycoproteins has been significantly improved. From quantified intact glycopeptides, the difference of protein glycosylation at multiple levels, e.g., glycoprotein, glycan, glycosite, and site-specific glycans, could be obtained for different samples. However, the manual analysis of the intact glycopeptide quantitative data at multiple levels is tedious and time consuming. In this study, we have developed a software tool named "GP-Marker" to facilitate large-scale data mining of spectra dataset of intact N-glycopeptide at multiple levels. This software provides a user-friendly and interactive interface, offering operational tools for machine learning to researchers without programming backgrounds. It includes a range of visualization plots displaying differential glycosylation and provides the ability to extract multi-level data analysis from intact glycopeptide data quantified by Glyco-Decipher.

O-Search-Pattern: A Searching Tool Utilizing the Y-Ion Pattern to Enhance O-Glycopeptide Identification for the Analysis of O-GalNAc Glycoproteome.

Different from N-linked glycosylation, the core structures of mucin type O-glycans are much more diverse, and the sensitive interpretation of O-glycopeptide spectra remains a challenge. The Y-ion pattern, a series of Y-ions with known mass gaps derived from the penta-saccharide core structure of N-linked glycosylation, is exploited to facilitate N-glycopeptide identification from their spectra. However, the pattern of Y ions in O-glycopeptides has not been well studied. In this study, we found that the Y-ion patterns were also frequently observed in the spectra of O-glycopeptides, and a special search approach is presented to identify O-glycopeptides by utilizing the Y-ion patterns. In this strategy, theoretical O-glycan Y-ion patterns are constructed to match the experimental Y-ions in O-glycopeptide spectra, which enables the determination of the mass of some glycans and results in the reduction of searching space. In addition, a Y-ion pattern-based deisotope process is also developed to correct the precursor m/z. The new search strategy was applied to search a human serum data set, and 15.4%-199.0% more O-glycopeptide-spectrum matches (OGPSMs) and 19.6%-107.1% more glycopeptide sequence identifications than other state-of-the-art software tools were observed. This search mode, the O-Search-Pattern, has been implemented into our database search software, MS-Decipher, and is recommended for searching the O-glycopeptide spectra acquired by sceHCD (stepped collision energy higher-energy collisional dissociation).

Development of an Integrated Platform for the Simultaneous Enrichment and Characterization of N- and O-Linked Intact Glycopeptides.

Both N-linked glycosylation and O-linked glycosylation play essential roles in the onset and progression of various diseases including cancer, and N-/O-linked site-specific glycans have been proven to be promising biomarkers for the discrimination of cancer. However, the micro-heterogeneity and low abundance nature of N-/O-linked glycosylation, as well as the time-consuming and tedious procedures for the enrichment of O-linked intact glycopeptides, pose great challenges for their efficient and accurate characterization. In this study, we developed an integrated platform for the simultaneous enrichment and characterization of N- and O-linked intact glycopeptides from the same serum sample. By fine-tuning the experimental conditions, we demonstrated that this platform allowed the selective separation of N- and O-linked intact glycopeptides into two fractions, with 85.1% O-linked intact glycopeptides presented in the first fraction and 93.4% N-linked intact glycopeptides presented in the second fraction. Determined with high reproducibility, this platform was further applied to the differential analysis of serum samples of gastric cancer and health control, which revealed 17 and 181 significantly changed O-linked and N-linked intact glycopeptides. Interestingly, five glycoproteins containing both significant regulation of N- and O-glycosylation were observed, hinting potential co-regulation of different types of glycosylation during tumor progress. In summary, this integrated platform opened a potentially useful avenue for the global analysis of protein glycosylation and can serve as a useful tool for the characterization of N-/O-linked intact glycopeptides at the proteomics scale.

O-Glycopeptide Truncation Strategy for Heterogeneous O-GalNAc Glycoproteomics Characterization.

Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 µL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.

MS-Decipher: a user-friendly proteome database search software with an emphasis on deciphering the spectra of O-linked glycopeptides.

MOTIVATION: The interpretation of mass spectrometry (MS) data is a crucial step in proteomics analysis, and the identification of post-translational modifications (PTMs) is vital for the understanding of the regulation mechanism of the living system. Among various PTMs, glycosylation is one of the most diverse ones. Though many search engines have been developed to decipher proteomic data, some of them are difficult to operate and have poor performance on glycoproteomic datasets compared to advanced glycoproteomic software. RESULTS: To simplify the analysis of proteomic datasets, especially O-glycoproteomic datasets, here, we present a user-friendly proteomic database search platform, MS-Decipher, for the identification of peptides from MS data. Two scoring schemes can be chosen for peptide-spectra matching. It was found that MS-Decipher had the same sensitivity and confidence in peptide identification compared to traditional database searching software. In addition, a special search mode, O-Search, is integrated into MS-Decipher to identify O-glycopeptides for O-glycoproteomic analysis. Compared with Mascot, MetaMorpheus and MSFragger, MS-Decipher can obtain about 139.9%, 48.8% and 6.9% more O-glycopeptide-spectrum matches. A useful tool is provided in MS-Decipher for the visualization of O-glycopeptide-spectra matches. MS-Decipher has a user-friendly graphical user interface, making it easier to operate. Several file formats are available in the searching and validation steps. MS-Decipher is implemented with Java, and can be used cross-platform. AVAILABILITY AND IMPLEMENTATION: MS-Decipher is freely available at https://github.com/DICP-1809/MS-Decipher for academic use. For detailed implementation steps, please see the user guide. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptomic and functional analysis provides molecular insights into multicellular trichome development.

Trichomes, the hair-like structures located on aerial parts of most vascular plants, are associated with a wide array of biological processes and affect the economic value of certain species. The processes involved in unicellular trichome formation have been well-studied in Arabidopsis (Arabidopsis thaliana). However, our understanding of the morphological changes and the underlying molecular processes involved in multicellular trichome development is limited. Here, we studied the dynamic developmental processes involved in glandular and nonglandular multicellular trichome formation in cucumber (Cucumis sativus L.) and divided these processes into five sequential stages. To gain insights into the underlying mechanisms of multicellular trichome formation, we performed a time-course transcriptome analysis using RNA-sequencing analysis. A total of 711 multicellular trichome-related genes were screened and a model for multicellular trichome formation was developed. The transcriptome and co-expression datasets were validated by reverse transcription-quantitative PCR and in situ hybridization. In addition, virus-induced gene silencing analysis revealed that CsHOMEOBOX3 (CsHOX3) and CsbHLH1 are involved in nonglandular trichome elongation and glandular trichome formation, respectively, which corresponds with the transcriptome data. This study presents a transcriptome atlas that provides insights into the molecular processes involved in multicellular trichome formation in cucumber and can be an important resource for future functional studies.

Risk Factors for Postoperative Knee Stiffness in Patients with Anteromedial Knee Osteoarthritis Undergoing Unicompartmental Knee Arthroplasty with Cemented Prostheses: A Short-Term, Retrospective, Case-Control Study.

BACKGROUND The present study was performed to determine the potential risk factors for postoperative knee stiffness in patients with anteromedial knee osteoarthritis undergoing unicompartmental knee arthroplasty with cemented prostheses. MATERIAL AND METHODS This retrospective cohort study evaluated patients with anteromedial knee osteoarthritis who underwent medial unicompartmental knee arthroplasty at our hospital between May 2017 and May 2020. The patients were divided into 2 groups according to their prognosis: those who experienced knee stiffness after undergoing unicompartmental knee arthroplasty and those who did not. The factors associated with stiffness after UKA were identified using univariate analysis. Frequencies are used to express categorical variables, while mean±SD is used to express continuous variables. The t test and chi-square test were used. A multivariate logistic regression model was built to identify the risk factors for postoperative stiffness. RESULTS We included 590 knees in the study after unicompartmental knee arthroplasty. The overall incidence of postoperative stiffness in unicompartmental knee arthroplasty surgery was 10.17%. In terms of the radiological measurements, varus deformity (70.34% vs 29.66%) and tibial component posterior slope angle (4.8±2.0 vs 4.6±2.0, P<0.001) were significantly differences between the 2 groups. Four independent risk factors for stiffness after unicompartmental knee arthroplasty were identified: age (95% CI, 1.022-1.048), varus deformity (95% CI, 1.186-1.192), tibial component posterior slope angle (95% CI, 0.550-0.870), and preoperative maximum flexion (95% CI, 0.896-0.923). CONCLUSIONS The overall incidence of postoperative knee stiffness in patients with anteromedial knee osteoarthritis undergoing unicompartmental knee arthroplasty with cemented prostheses was 10.17%, which was at a moderate level compared to patients with other diseases undergoing unicompartmental knee arthroplasty. Four independent risk factors were identified: age, varus deformity, preoperative maximum flexion, and tibial component posterior slope angle. Awareness these risk factors might help surgeons prevent the occurrence of postoperative knee stiffness in patients with UKA.

Identification and Functional Characterization of CsMYCs in Cucumber Glandular Trichome Development.

Glandular trichomes (GTs), specialized structures formed by the differentiation of plant epidermal cells, are known to play important roles in the resistance of plants to external biotic and abiotic stresses. These structures are capable of storing and secreting secondary metabolites, which often have important agricultural and medicinal values. In order to better understand the molecular developmental mechanisms of GTs, studies have been conducted in a variety of crops, including tomato (Solanum lycopersicum), sweetworm (Artemisia annua), and cotton (Gossypium hirsutum). The MYC transcription factor of the basic helix-loop-helix (bHLH) transcription factor family has been found to play an important role in GT development. In this study, a total of 13 cucumber MYC transcription factors were identified in the cucumber (Cucumis sativus L.) genome. After performing phylogenetic analyses and conserved motifs on the 13 CsMYCs in comparison to previously reported MYC transcription factors that regulate trichome development, seven candidate MYC transcription factors were selected. Through virus-induced gene silencing (VIGS), CsMYC2 is found to negatively regulate GT formation while CsMYC4, CsMYC5, CsMYC6, CsMYC7, and CsMYC8 are found to positively regulate GT formation. Furthermore, the two master effector genes, CsMYC2 and CsMYC7, are observed to have similar expression patterns indicating that they co-regulate the balance of GT development in an antagonistic way.

TINY BRANCHED HAIR functions in multicellular trichome development through an ethylene pathway in Cucumis sativus L.

The fruit trichomes of Cucurbitaceae are widely desired in many Asian countries and have been a key determinant of cucumber (Cucumis sativus L.) cultivar selection for commercial production and breeding. However, our understanding of the initiation and development of cucumber trichomes is still limited. Here, we found that the cucumber TINY BRANCHED HAIR (TBH) gene is preferentially expressed in multicellular trichomes. Overexpression of CsTBH in tbh mutants restored the trichome phenotype and increased the percentage of female flowers, whereas silencing of CsTBH in wild-type plants resulted in stunted trichomes with a lower rate of female flowers. Furthermore, we provide evidence that CsTBH can directly bind to the promoters of cucumber 1-Aminocyclopropane-1-Carboxylate Synthase (CsACS) genes and regulate their expression, which affects multicellular trichome development, ethylene accumulation, and sex expression. Two cucumber acs mutants with different trichome morphology and sex morphs compared with their near-isogenic line further support our findings. Collectively, our study provides new information on the molecular mechanism of CsTBH in regulating multicellular trichome development and sex expression through an ethylene pathway.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Development of Parallel Reaction Monitoring Assays for the Detection of Aggressive Prostate Cancer Using Urinary Glycoproteins.

Recently, we have found that two urinary glycoproteins, prostatic acid phosphatase (ACPP) and clusterin (CLU), combined with serum prostate-specific antigen (PSA) can serve as a three-signature panel for detecting aggressive prostate cancer (PCa) based on a quantitative glycoproteomic study. To facilitate the translation of candidates into clinically applicable tests, robust and accurate targeted parallel reaction monitoring (PRM) assays that can be widely adopted in multiple labs were developed in this study. The developed PRM assays for the urinary glycopeptides, FLN*ESYK from ACPP and EDALN*ETR from CLU, demonstrated good repeatability and a sufficient working range covering three to four orders of magnitude, and their performance in differentiating aggressive PCa was assessed by the quantitative analysis of urine specimens collected from 69 nonaggressive (Gleason score = 6) and 73 aggressive (Gleason ≥ 8) PCa patients. When ACPP combined with CLU, the discrimination power was improved from an area under a curve (AUC) of 0.66 to 0.78. By combining ACPP, CLU, and serum PSA to form a three-signature panel, the AUC was further improved to 0.83 (sensitivity: 84.9%, specificity: 66.7%). Since the serum PSA test alone had an AUC of 0.68, our results demonstrated that the new urinary glycopeptide PRM assays can serve as an adjunct to the serum PSA test to achieve better predictive power toward aggressive PCa. In summary, our developed PRM assays for urinary glycopeptides were successfully applied to clinical PCa urine samples with a promising performance in aggressive PCa detection.

Data-Independent Acquisition-Based Mass Spectrometry (DIA-MS) for Quantitative Analysis of Intact N-Linked Glycopeptides.

N-linked protein glycosylation is a key regulator in various biological functions. Previous studies have shown that aberrant glycosylation is associated with many diseases. Therefore, it is essential to elucidate protein modifications of glycosylation by quantitatively profiling intact N-linked glycopeptides. Data-independent acquisition (DIA) mass spectrometry (MS) is a cost-effective, flexible, and high-throughput method for global proteomics. However, substantial challenges are still present in the quantitative analysis of intact glycopeptides with high accuracy at high throughput. In this study, we have established a novel integrated platform for the DIA analysis of intact glycopeptides isolated from complex samples. The established analysis platform utilizes a well-designed DIA-MS method for raw data collection, a spectral library constructed specifically for intact glycopeptide quantification providing accurate results by the inclusion of Y ions for quantification and filtering of quantified intact glycopeptides with low-quality MS2 spectra automatically using a set of criteria. Intact glycopeptides isolated from human serum were used to evaluate the performance of the integrated platform. By utilizing 100 isolation windows for DIA data acquisition, a well-constructed human serum spectral library containing 1123 nonredundant intact glycopeptides with Y ions, and automated data inspection, 620 intact glycopeptides were quantified with high confidence from DIA-MS. In summary, our integrated platform can serve as a reliable quantitative tool for characterizing intact glycopeptides isolated from complex biological samples to assist our understanding of biological functions of N-linked glycosylation.

The impact of global dimming on crop yields is determined by the source-sink imbalance of carbon during grain filling.

Global dimming reduces incident global radiation but increases the fraction of diffuse radiation, and thus affects crop yields; however, the underlying mechanisms of such an effect have not been revealed. We hypothesized that crop source-sink imbalance of either carbon (C) or nitrogen (N) during grain filling is a key factor underlying the effect of global dimming on yields. We presented a practical framework to assess both C and N source-sink relationships, using data of biomass and N accumulation from periodical sampling conducted in field experiments for wheat and rice from 2013 to 2016. We found a fertilization effect of the increased diffuse radiation fraction under global dimming, which alleviated the negative impact of decreased global radiation on source supply and sink growth, but the source supply and sink growth were still decreased by dimming, for both C and N. In wheat, the C source supply decreased more than the C sink demand, and as a result, crops remobilized more pre-heading C reserves, in response to dimming. However, these responses were converse in rice, which presumably stemmed from the more increment in radiation use efficiency and the more limited sink size in rice than wheat. The global dimming affected source supply and sink growth of C more significantly than that of N. Therefore, yields in both crops were dependent more on the source-sink imbalance of C than that of N during grain filling. Our revealed source-sink relationships, and their differences and similarities between wheat and rice, provide a basis for designing strategies to alleviate the impact of global dimming on crop productivity.

Glycans, Glycosite, and Intact Glycopeptide Analysis of N-Linked Glycoproteins Using Liquid Handling Systems.

Aberrant glycosylation has been shown to associate with disease progression, and with glycoproteins representing the major protein component of biological fluids this makes them attractive targets for disease monitoring. Leveraging glycoproteomic analysis via mass spectrometry (MS) could provide the insight into the altered glycosylation patterns that relate to disease progression. However, investigation of large sample cohorts requires rapid, efficient, and highly reproducible sample preparation. To address the limitation, we developed a high-throughput method for characterizing glycans, glycosites, and intact glycopeptides (IGPs) derived from N-linked glycoproteins. We combined disparate peptide enrichment strategies (i.e., hydrophilic and hydrophobic) and a liquid handling platform allowing for a high throughput and rapid enrichment of IGP in a 96-well plate format. The C18/MAX-Tip workflow reduced sample processing time and facilitated the selective enrichment of IGPs from complex samples. Furthermore, our approach enabled the analysis of deglycosylated peptides and glycans from enriched IGPs following PNGase F digest. Following development and optimization of the C18/MAX-Tip methodology using the standard glycoprotein, fetuin, we investigated normal urine samples to obtain N-linked glycoprotein information. Together, our method enables a high-throughput enrichment of glycan, glycosites, and IGPs from biological samples.

The HD-ZIP IV transcription factor GL2-LIKE regulates male flowering time and fertility in cucumber.

Cucumber is dioecious by nature, having both male and female flowers, and is a model system for unisexual flower development. Knowledge related to male flowering is limited, but it is reported to be regulated by transcription factors and hormone signals. Here, we report functional characterization of the cucumber (Cucumis sativus) GL2-LIKE gene, which encodes a homeodomain leucine zipper (HD-ZIP) IV transcription factor that plays an important role in regulating male flower development. Spatial-temporal expression analyses revealed high-level expression of CsGL2-LIKE in the male flower buds and anthers. CsGL2-LIKE is closely related to AtGL2, which is known to play a key role in trichome development. However, ectopic expression of CsGL2-LIKE in Arabidopsis gl2-8 mutant was unable to rescue the gl2-8 phenotype. Interestingly, the silencing of CsGL2-LIKE delayed male flowering by inhibiting the expression of the florigen gene FT and reduced pollen vigor and seed viability. Protein-protein interaction assays showed that CsGL2-LIKE interacts with the jasmonate ZIM domain protein CsJAZ1 to form a HD-ZIP IV-CsJAZ1 complex. Collectively, our study indicates that CsGL2-LIKE regulates male flowering in cucumber, and reveals a novel function of a HD-ZIP IV transcription factor in regulating male flower development of cucumber.

Wernekink commissure syndrome secondary to a rare 'V'-shaped pure midbrain infarction: a case report and review of the literature.

Purpose: Wernekink commissure syndrome is a typical but extremely rare mesencephalic syndrome, and generally presents with bilateral cerebellar dysfunction, diverse oculomotor disorders and occasionally delayed-onset palatal myoclonus or tremor. However, it has been reported infrequently.Methods: We report a case of a 55-year-old man who suffered an acute paramedian midbrain infarction presenting with bilateral cerebellar ataxia, bilateral anterior internuclear ophthalmoplegia and unilateral pseudoabducens palsy, which is confirmed as Wernekink commissure syndrome by magnetic resonance imaging (MRI). We summarized the clinical data of this entity and performed a literature review of 20 previous reports of patients with this syndrome.Results: In combination with previous reports, we found that the most common symptom was bilateral cerebellar ataxia (100%) and other frequent symptoms were oculomotor disorders (81%), delayed palatal myoclonus or tremor (33%) and consciousness dysfunction (33%). Lesions on brain MRI of all patients affected the area of caudal paramedian midbrain (CPM).Conclusion: Bilateral cerebellar ataxia and lesions involved in the area of CPM on MRI are the major features of Wernekink commissure syndrome and should be the necessary conditions of diagnostic criteria. The simultaneous occurrence of bilateral cerebellar ataxia and oculomotor disorders is significant for localization diagnosis. Consciousness dysfunction is also a relatively frequent symptom of this syndrome. Moreover, pseudoabducens palsy might be attributed to a midbrain lesion. Clinicians should be familiar with and early to recognize this unique syndrome to avoid misdiagnosis.

Intravenous thrombolysis for pure pontine infarcts caused by cardiac myxoma: a case report and literature review.

Purpose: Cardiac myxoma (CM) is a rare but important cause of ischemic stroke, and typically involves the middle cerebral artery and rarely affects the brainstem only. The safety and efficacy of intravenous thrombolysis (IVT) for CM-related acute cerebral embolism are not clear.Methods: We report a case of a 55-year-old woman who suffered a CM-related acute cerebral embolism presented with pure pontine infarcts and achieved a favorable prognosis by IVT with urokinase. We summarized the clinical data of this entity and performed a literature review of 21 previous reports of patients with CM-related acute cerebral embolism who were treated with IVT.Results: In combination with previous reports, we found that the majority of patients (81.8%) obtained improvements in symptoms after IVT, including 63.6% in remarkable clinical improvement. The total rate of IVT-induced intracerebral hemorrhage was 22.7% and all occurred within 36 h, including hemorrhagic infarction type 1 (4.5%) and parenchymal hematoma type 2 (18.2%). Most of the cases had relatively good outcomes and no case died due to IVT.Conclusion: Taken together, our findings support the use of IVT as an effective and safe tool for the ultra-early treatment of CM-related acute phase ischemic stroke.