Human cytomegalovirus pUL97 upregulates SOCS3 expression via transcription factor RFX7 in neural progenitor cells.

Congenital human cytomegalovirus (HCMV) infection causes severe damage to the fetal brain, and the underlying mechanisms remain elusive. Cytokine signaling is delicately controlled in the fetal central nervous system to ensure proper development. Here we show that suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the IL-6 cytokine family signaling, was upregulated during HCMV infection in primary neural progenitor cells (NPCs) with a biphasic expression pattern. From viral protein screening, pUL97 emerged as the viral factor responsible for prolonged SOCS3 upregulation. Further, by proteomic analysis of the pUL97-interacting host proteins, regulatory factor X 7 (RFX7) was identified as the transcription factor responsible for the regulation. Depletion of either pUL97 or RFX7 prevented the HCMV-induced SOCS3 upregulation in NPCs. With a promoter-luciferase activity assay, we demonstrated that the pUL97 kinase activity and RFX7 were required for SOCS3 upregulation. Moreover, the RFX7 phosphorylation level was increased by either UL97-expressing or HCMV-infection in NPCs, suggesting that pUL97 induces RFX7 phosphorylation to drive SOCS3 transcription. We further revealed that elevated SOCS3 expression impaired NPC proliferation and migration in vitro and caused NPCs migration defects in vivo. Taken together, these findings uncover a novel regulatory mechanism of sustained SOCS3 expression in HCMV-infected NPCs, which perturbs IL-6 cytokine family signaling, leads to NPCs proliferation and migration defects, and consequently affects fetal brain development.

Human Cytomegalovirus IE1 Impairs Neuronal Migration by Downregulating Connexin 43.

Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Mutations in BrABCG26, encoding an ATP-binding cassette transporter, are responsible for male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: The gene BrABCG26 responsible for male sterility of Chinese cabbage was confirmed by two allelic mutants. Male-sterile lines are an important way of heterosis utilization in Chinese cabbage. In this study, two allelic male-sterile mutants msm3-1 and msm3-2 were obtained from a Chinese cabbage double haploid (DH) line 'FT' by using EMS-mutagenesis. Compared to the wild-type 'FT,' the stamens of mutants were completely degenerated and had no pollen, and other characters had no obvious differences. Cytological observation revealed that the failure of vacuolation of the mononuclear microspore, accompanied by abnormal tapetal degradation, resulted in anther abortion in mutants. Genetic analysis showed that a recessive gene controlled the mutant trait. MutMap combined with kompetitive allele specific PCR genotyping analyses showed that BraA01g038270.3C, encoding a transporter ABCG26 that played a vital role in pollen wall formation, was the candidate gene for msm3-1, named BrABCG26. Compared with wild-type 'FT,' the mutations existed on the second exon (C to T) and the sixth exon (C to T) of BrABCG26 gene in mutants msm3-1 and msm3-2, leading to the loss-of-function truncated protein, which verified the BrABCG26 function in stamen development. Subcellular localization and expression pattern analysis indicated that BrABCG26 was localized in the nucleus and was expressed in all organs, with the highest expression in flower buds. Compared to the wild-type 'FT,' the expressions of BrABCG26 were significantly reduced in flower buds and anthers of mutants. Promoter activity analysis showed that a strong GUS signal was detected in flower buds. These results indicated that BrABCG26 is responsible for the male sterility of msm3 mutants in Chinese cabbage.

pH/GSH dual-responsive nanoparticle for auto-amplified tumor therapy of breast cancer.

Breast cancer remains a malignancy that poses a serious threat to human health worldwide. Chemotherapy is one of the most widely effective cancer treatments in clinical practice, but it has some drawbacks such as poor targeting, high toxicity, numerous side effects, and susceptibility to drug resistance. For auto-amplified tumor therapy, a nanoparticle designated GDTF is prepared by wrapping gambogic acid (GA)-loaded dendritic porous silica nanoparticles (DPSNs) with a tannic acid (TA)-Fe(III) coating layer. GDTF possesses the properties of near-infrared (NIR)-enhanced and pH/glutathione (GSH) dual-responsive drug release, photothermal conversion, GSH depletion and hydroxyl radical (·OH) production. When GDTF is exposed to NIR laser irradiation, it can effectively inhibit cell proliferation and tumor growth both in vitro and in vivo with limited toxicity. This may be due to the synergistic effect of enhanced tumor accumulation, and elevated reactive oxygen species (ROS) production, GSH depletion, and TrxR activity reduction. This study highlights the enormous potential of auto-amplified tumor therapy.

iTRAQ-Based Proteomics Analysis of Human Cytomegalovirus Latency and Reactivation in T98G Cells.

Human cytomegalovirus (HCMV) establishes a persistent/latent infection after primary infection, and the host factor(s) plays a key role in regulating HCMV infection status. The spread of reactivated HCMV via the hematogenous or neural route usually results in severe diseases in newborns and immunocompromised individuals. As the primary reservoirs in vivo, cells of myeloid lineage have been utilized extensively to study HCMV infection. However, the molecular mechanism of HCMV latency/reactivation in neural cells is still poorly understood. We previously showed that HCMV-infected T98G cells maintain a large number of viral genomes and support HCMV reactivation from latency upon cAMP/IBMX treatment. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics to characterize cellular protein changes during HCMV latency and reactivation in T98G cells. A total of 168 differentially expressed proteins (DEPs) were identified, including 89 proteins in latency and 85 proteins in reactivation. Bioinformatics analysis showed that a few biological pathways were associated with HCMV latency or reactivation. Moreover, we validated 16 DEPs by both mRNA and protein expression profiles and further evaluated the effects of ApoE and the phosphatidylinositol 3-kinase (PI3K) pathway on HCMV infection. ApoE knockdown reduced HCMV loads and virus release, whereas overexpressing ApoE hampered HCMV latent infection, indicating a role in HCMV latency establishment/maintenance. Blocking the PI3K pathway by LY294002, a PI3K inhibitor, induced HCMV reactivation from latency in T98G cells. Overall, this comparative proteomics analysis delineates the cellular protein changes during HCMV latency and reactivation and provides a road map to advance our understanding of the mechanism(s) in the context of neural cells. IMPORTANCE Human cytomegalovirus (HCMV) is a highly transmissible betaherpesvirus that has a prevalence of 60% to 90% worldwide. This opportunist pathogen poses a significant threat to newborns and immunosuppressed individuals. One major obstacle for developing effective therapeutics is a poor understanding of HCMV latency/reactivation mechanisms. This study presents, for the first time, a systemic analysis of host cell protein expression changes during HCMV latency establishment and reactivation processes in neural cells. We showed that ApoE was downregulated by HCMV to facilitate latent infection. Also, the proteomics analysis has associated a few PI3K pathway-related proteins with HCMV reactivation. Altogether, this study highlights multiple host proteins and signaling pathways that can be further investigated as potential druggable targets for HCMV-related diseases, especially brain disorders.

Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis.

Human cytomegalovirus (HCMV) has a large (∼235 kb) genome with more than 200 predicted open reading frames that exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here, we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was upregulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and colocalized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. IMPORTANCE During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for antiviral treatment.

Role of Brassica orphan gene BrLFM on leafy head formation in Chinese cabbage (Brassica rapa).

KEY MESSAGE: Brassica orphan gene BrFLM, identified by two allelic mutants, was involved in leafy head formation in Chinese cabbage. Leafy head formation is a unique agronomic trait of Chinese cabbage that determines its yield and quality. In our previous study, an EMS mutagenesis Chinese cabbage mutant library was constructed using the heading Chinese cabbage double haploid (DH) line FT as the wild-type. Here, we screened two extremely similar leafy head deficiency mutants lfm-1 and lfm-2 with geotropic growth leaves from the library to investigate the gene(s) related to leafy head formation. Reciprocal crossing results showed that these two mutants were allelic. We utilized lfm-1 to identify the mutant gene(s). Genetic analysis showed that the mutated trait was controlled by a single nuclear gene Brlfm. Mutmap analysis showed that Brlfm was located on chromosome A05, and BraA05g012440.3C or BraA05g021450.3C were the candidate gene. Kompetitive allele-specific PCR analysis eliminated BraA05g012440.3C from the candidates. Sanger sequencing identified an SNP from G to A at the 271st nucleotide on BraA05g021450.3C. The sequencing of lfm-2 detected another non-synonymous SNP (G to A) located at the 266st nucleotide on BraA05g021450.3C, which verified its function on leafy head formation. We blasted BraA05g021450.3C on database and found that it belongs to a Brassica orphan gene encoding an unknown 13.74 kDa protein, named BrLFM. Subcellular localization showed that BrLFM was located in the nucleus. These findings reveal that BrLFM is involved in leafy head formation in Chinese cabbage.

BrACOS5 mutations induced male sterility via impeding pollen exine formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: BrACOS5 mutations led to male sterility of Chinese cabbage verified in three allelic male-sterile mutants. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major vegetable crops in East Asia, and the utilization of male-sterile line is an important measure for its hybrid seed production. Herein, we isolated three allelic male-sterile mutants, msm1-1, msm1-2 and msm1-3, from an ethyl methane sulfonate (EMS) mutagenized population of Chinese cabbage double-haploid (DH) line 'FT', whose microspores were completely aborted with severely absent exine, and tapetums were abnormally developed. Genetic analyses indicated that the three male-sterile mutants belonged to allelic mutation and were triggered by the same recessive nuclear gene. MutMap-based gene mapping and kompetitive allele-specific PCR (KASP) analysis demonstrated that three different single-nucleotide polymorphisms (SNPs) of BraA09g012710.3C were responsible for the male sterility of msm1-1/2/3, respectively. BraA09g012710.3C is orthologous of Arabidopsis thaliana ACOS5 (AT1G62940), encoding an acyl-CoA synthetase in sporopollenin biosynthesis, and specifically expressed in anther, so we named BraA09g012710.3C as BrACOS5. BrACOS5 localizes to the endoplasmic reticulum (ER). Mutations of BrACOS5 resulted in decreased enzyme activities and altered fatty acid contents in msm1 anthers. As well as the transcript accumulations of putative orthologs involved in sporopollenin biosynthesis were significantly down-regulated excluding BrPKSA. These results provide strong evidence for the integral role of BrACOS5 in conserved sporopollenin biosynthesis pathway and also contribute to uncovering exine development pattern and underlying male sterility mechanism in Chinese cabbage.

Implementation fidelity to a behavioral diabetes prevention intervention in two New York City safety net primary care practices.

BACKGROUND: It is critical to assess implementation fidelity of evidence-based interventions and factors moderating fidelity, to understand the reasons for their success or failure. However, fidelity and fidelity moderators are seldom systematically reported. The study objective was to conduct a concurrent implementation fidelity evaluation and examine fidelity moderators of CHORD (Community Health Outreach to Reduce Diabetes), a pragmatic, cluster-randomized, controlled trial to test the impact of a Community Health Workers (CHW)-led health coaching intervention to prevent incident type 2 Diabetes Mellitus in New York (NY). METHODS: We applied the Conceptual Framework for Implementation Fidelity to assess implementation fidelity and factors moderating it across the four core intervention components: patient goal setting, education topic coaching, primary care (PC) visits, and referrals to address social determinants of health (SDH), using descriptive statistics and regression models. PC patients with prediabetes receiving care from safety-net patient-centered medical homes (PCMHs) at either, VA NY Harbor or at Bellevue Hospital (BH) were eligible to be randomized into the CHW-led CHORD intervention or usual care. Among 559 patients randomized and enrolled in the intervention group, 79.4% completed the intake survey and were included in the analytic sample for fidelity assessment. Fidelity was measured as coverage, content adherence and frequency of each core component, and the moderators assessed were implementation site and patient activation measure. RESULTS: Content adherence was high for three components with nearly 80.0% of patients setting ≥ 1 goal, having ≥ 1 PC visit and receiving ≥ 1 education session. Only 45.0% patients received ≥ 1 SDH referral. After adjusting for patient gender, language, race, ethnicity, and age, the implementation site moderated adherence to goal setting (77.4% BH vs. 87.7% VA), educational coaching (78.9% BH vs. 88.3% VA), number of successful CHW-patient encounters (6 BH vs 4 VA) and percent of patients receiving all four components (41.1% BH vs. 25.7% VA). CONCLUSIONS: The fidelity to the four CHORD intervention components differed between the two implementation sites, demonstrating the challenges in implementing complex evidence-based interventions in different settings. Our findings underscore the importance of measuring implementation fidelity in contextualizing the outcomes of randomized trials of complex multi-site behavioral interventions. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov on 30/12/2016 and the registration number is NCT03006666 .

Rapid Adoption of Telemedicine Increases Opioid Prescribing in Orthopedic Surgery.

Background: The COVID-19 pandemic led to health care practitioners utilizing new technologies to deliver health care, including telemedicine. The purpose of this study was to examine the effect of rapidly proliferative use of video visits on opioid prescribing to orthopedic patients at a large academic health system that had existing procedure-specific opioid prescribing guidelines. Methods: This IRB-exempt study examined 651 opioid prescriptions written to patients who had video (visual and audio), telephone (audio only), or in-person encounters at our institution from March 1 to June 1, 2020 and compared them with 963 prescriptions written during the same months in 2019. Prescriptions were converted into daily milligram morphine equivalents (MMEs) to facilitate direct comparison. Chi-square testing was used to compare categorical data, whereas analysis of variance and Mann-Whitney tests were used to compare numerical data between groups. Statistical significance was set at <0.05. Results: Six hundred fifty-one of 1,614 prescriptions analyzed (40.3%) occurred during the pandemic. Patients prescribed opioids during video visits were prescribed 53.3 ± 37 MME, significantly higher than in-person (p = 0.002) or audio visits (p < 0.001) before or during the pandemic. Prepandemic, significantly higher MME were prescribed for in-person versus audio only visits (41.6 ± 89 vs. 30.2 ± 28 MME; p = 0.026); during the pandemic, there was no difference between these groups (p = 0.91). Significantly higher MME were prescribed by Nurse Practitioners and Physician Associates versus MD or DO prescribers for both time periods (51.3 ± 109 vs. 27.9 ± 42 MME; p < 0.001; 42.9 ± 70 vs. 28.2 ± 42 MME; p < 0.001). Conclusion: During crisis and with new technology, we should be vigilant about prescribing of opioid analgesics. Despite well-established protocols, patients received significantly higher MME through video than for other encounter types, including in-person encounters. In addition, significantly higher MME were prescribed by mid-level prescribers compared with DOs or MDs. Institutions should ensure these prescribers are involved during creation of opioid prescribing protocols after orthopedic surgery.

BrCWM Mutation Disrupted Leaf Flattening in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Leaf flattening plays a vital role in the establishment of plant architecture, which is closely related to plant photosynthesis and, thus, influences the product yield and quality of Chinese cabbage. In this study, we used the doubled haploid line 'FT' of Chinese cabbage as the wild type for ethyl methanesulfonate (EMS) mutagenesis and obtained a mutant cwm with stably inherited compact and wrinkled leaves. Genetic analysis revealed that the mutated trait was controlled by a single recessive nuclear gene, Brcwm. Brcwm was preliminarily mapped to chromosome A07 based on bulked segregant RNA sequencing (BSR-seq) and fine-mapped to a 205.66 kb region containing 39 genes between Indel12 and Indel21 using SSR and Indel analysis. According to the whole-genome re-sequencing results, we found that there was only one nonsynonymous single nucleotide polymorphism (SNP) (C to T) within the target interval on exon 4 of BraA07g021970.3C, which resulted in a proline to serine amino acid substitution. The mutated trait co-segregated with the SNP. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that BraA07g021970.3C expression was dramatically higher in 'FT' leaves than that in cwm leaves. BraA07g021970.3C is homologous to AT3G55000 encoding a protein related to cortical microtubule organization. A similar phenotype of dwarfism and wrinkled leaves was observed in the recessive homozygous mutant cwm-f1 of AT3G55000, and its T3 transgenic lines were restored to the Arabidopsis wild-type phenotype through ectopic overexpression of BraA07g021970.3C. These results verified that BraA07g021970.3C was the target gene essential for leaf flattening in Chinese cabbage.

Wrist angle measurements in predicting the presence of a displaced scaphoid fracture.

PURPOSE: Early diagnosis and treatment of scaphoid fractures is critical to achieve union and prevent wrist degenerative arthritis. Plain radiographic measurements can guide the early approach to suspected fractures. Specifically, the ability to predict fracture or fracture stability from plain radiographs may allow the traumatologist to expand initial referral to three-dimensional imaging. We evaluated the ability of four measured angles to predict presence of a scaphoid fracture and stability. METHODS: Fifty patients with a scaphoid fracture and 50 patients without fracture were evaluated for the cortical ring sign, scapho-lunate gapping, lateral scapholunate (SL), radio-scaphoid (RS), radio-lunate (RL), and radio-capitate (RC) angles by two-blinded observers. RESULTS: Measurement of an increased SL interval was associated with the presence of a scaphoid fracture as diagnosed on three-dimensional imaging [odds ratio (OR) 3.0, confidence interval (CI) 1.53-5.87, p = < 0.01]. The measured RL angle was associated with fracture displacement (OR 1.13, 95% CI 1.02-1.25, p = 0.02). CONCLUSIONS: Scapholunate gapping on plain radiographs in the context of a clinically suspected scaphoid fracture should increase suspicion for a fracture and may prompt earlier 3-dimensional imaging, while the presence of an abnormal radiolunate angle should increase wariness for instability and can be used in preoperative planning.

Proteomic analysis of a plastid gene encoding RPS4 mutant in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

Plastids are important plant cell organelles containing a genome and bacterial-type 70S ribosomes-primarily composed of plastid ribosomal proteins and ribosomal RNAs. In this study, a chlorophyll-deficient mutant (cdm) obtained from double-haploid Chinese cabbage 'FT' was identified as a plastome mutant with an A-to-C base substitution in the plastid gene encoding the ribosomal protein RPS4. To further elucidate the function and regulatory mechanisms of RPS4, a comparative proteomic analysis was conducted between cdm and its wild-type 'FT' plants by isobaric tags and a relative and absolute quantitation (iTRAQ)-based strategy. A total of 6,245 proteins were identified, 540 of which were differentially abundant proteins (DAPs) in the leaves of cdm as compared to those of 'FT'-including 233 upregulated and 307 downregulated proteins. Upregulated DAPs were mainly involved in translation, organonitrogen compound biosynthetic process, ribosomes, and spliceosomes. Meanwhile, downregulated DAPs were mainly involved in photosynthesis, photosynthetic reaction centres, photosynthetic light harvesting, carbon fixation, and chlorophyll binding. These results indicated an important role of RPS4 in the regulation of growth and development of Chinese cabbage, possibly by regulating plastid translation activity by affecting the expression of specific photosynthesis- and cold stress-related proteins. Moreover, a multiple reaction monitoring (MRM) test and quantitative real-time polymerase chain reaction analysis confirmed our iTRAQ results. Quantitative proteomic analysis allowed us to confirm diverse changes in the metabolic pathways between cdm and 'FT' plants. This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in Chinese cabbage.

Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly.

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis.Importance Human cytomegalovirus (HCMV) has a large (∼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

MORC3 restricts human cytomegalovirus infection by suppressing the major immediate-early promoter activity.

During the long coevolution of human cytomegalovirus (HCMV) and humans, the host has formed a defense system of multiple layers to eradicate the invader, and the virus has developed various strategies to evade host surveillance programs. The intrinsic immunity primarily orchestrated by promyelocytic leukemia (PML) nuclear bodies (PML-NBs) represents the first line of defense against HCMV infection. Here, we demonstrate that microrchidia family CW-type zinc finger 3 (MORC3), a PML-NBs component, is a restriction factor targeting HCMV infection. We show that depletion of MORC3 through knockdown by RNA interference or knockout by CRISPR-Cas9 augmented immediate-early protein 1 (IE1) gene expression and subsequent viral replication, and overexpressing MORC3 inhibited HCMV replication by suppressing IE1 gene expression. To relief the restriction, HCMV induces transient reduction of MORC3 protein level via the ubiquitin-proteasome pathway during the immediate-early to early stage. However, MORC3 transcription is upregulated, and the protein level recovers in the late stages. Further analyses with temporal-controlled MORC3 expression and the major immediate-early promoter (MIEP)-based reporters show that MORC3 suppresses MIEP activity and consequent IE1 expression with the assistance of PML. Taken together, our data reveal that HCMV enforces temporary loss of MORC3 to evade its repression against the initiation of immediate-early gene expression.

Mutation in BrGGL7 gene encoding a GDSL esterase / lipase causes male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: MutMap and KASP analyses revealed that the BrGGL7 gene is responsible for the male-sterile trait of ftms1 in Chinese cabbage, with functional verification in Arabidopsis. The application of a male-sterile line is an ideal approach of hybrid seed production in Chinese cabbage. In this study, we obtained a male-sterile mutant (ftms1) from the double haploid line 'FT' using ethyl methane sulfonate (EMS) mutagenesis. The mutant was completely sterile due to abnormal enlargement and vacuolization of the tapetum cells. A single recessive nuclear gene was found to control male sterility in the mutant, while MutMap and KASP analyses identified BraA05g022470.3C (BrGGL7), which encodes a GDSL esterase / lipase, as the candidate mutant gene. A single nucleotide substitution from C to T occurred within the domain of BrGGL7 in ftms1, resulting in premature translation termination in the fourth exon. Meanwhile, qRT-PCR analysis indicated that BrGGL7 was prominently expressed in the anthers, and expression was greater in the wild-type 'FT' than ftms1. Genetic complementation of the orthologous Arabidopsis ggl7 mutant further confirmed the role of BrGGL7 in pollen development. These findings suggest that BrGGL7 plays a fundamental role in pollen formation, providing important insight into the molecular mechanisms underlying male sterility in Chinese cabbage.

BrKAO2 mutations disrupt leafy head formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: The role of BrKAO2 in leafy head formation was confirmed by using two allelic Chinese cabbage mutants. Chinese cabbage yield and quality are determined by leafy head formation. Cloning and characterising the key genes regulating leafy head formation are essential for its varietal improvement. We used an EMS-mutagenised population of the heading type 'FT' Chinese cabbage line and identified two allelic non-heading mutants, i.e. nhm3-1 and nhm3-2. Genetic analysis showed that the mutant trait was controlled by a single recessive gene. MutMap and Kompetitive Allele Specific PCR genotyping revealed that BraA05g012440.3C was the candidate gene, which encodes ent-kaurenoic acid oxidase 2 in gibberellin (GA) biosynthetic pathway. It was named BrKAO2. Two non-synonymous mutations in the second BrKAO2 exon, respectively, accounted for the mutant phenotypes of nhm3-1 and nhm3-2. BrKAO2 was expressed during all leaf development stages, and there were no significant differences between the wild type and mutants in terms of BrKAO2 expression. The mutant phenotypes were restored to the wild type via exogenous GA3 application. RNA-Seq was performed on wild-type 'FT', nhm3-1, and nhm3-1 + GA3 rosette leaves, and several key genes involved in GA biosynthesis, signal transduction, and leafy head development were identified. These findings indicate that BrKAO2 is responsible for the leafy head formation in nhm3 mutants.

Patient Acceptable Symptom State for the Forgotten Joint Score in Primary Total Knee Arthroplasty.

BACKGROUND: In order to better understand the clinical benefits of total knee arthroplasty (TKA) and improve the interpretability of the Forgotten Joint Score (FJS-12), the establishment of a meaningful change in score is necessary. The purpose of this study is to determine the threshold of the FJS-12 for detecting the patient acceptable symptom state (PASS) following primary TKA. METHODS: We retrospectively reviewed all patients who underwent elective, primary TKA and answered both the FJS-12 and the Knee Injury Osteoarthritis Outcome Survey, Joint Replacement KOOS, JR surveys 1-year postoperatively. The questionnaires were administered via a web-based electronic application. KOOS, JR score was used as the anchor. The anchor for PASS calculation should relate pain, physical function, and patient satisfaction. Two statistical methods were employed: (1) the receiver operating characteristic (ROC) curve point; (2) 75th percentile of the cumulative percentage curve of patients who had the KOOS, JR score difference larger than the cut-off value. RESULTS: This study included 457 patients. The mean 1-year FJS-12 score was 42.6 ± 27.8. The mean 1-year KOOS, JR score was 68.0 ± 17.2. A high positive correlation between FJS-12 and KOOS, JR was found (r = 0.72, P < .001) making the KOOS, JR a valid external anchor. The threshold score of the FJS-12 which maximized the sensitivity and specificity for detecting a PASS was 33.3 (AUC = 0.78, 95% CI [0.74, 0.83]). The cut-off value computed with the 75th percentile approach was 77.1 (95% CI [73.9, 81.5]). CONCLUSION: The PASS threshold for the FJS-12 was 33.3 and 77.1 at 1-year follow-up after primary TKA using the receiver operating characteristic (ROC) curve and 75th percentile approaches, respectively. These values can be used to assess the successful achievement of a forgotten joint. LEVEL III EVIDENCE: Retrospective Cohort Study.

Validation of Single-Outcome Questionnaire in Primary TKA and THA.

BACKGROUND: Patient-reported outcome measures can be used to evaluate post-operative health care quality and patient satisfaction. The Patient's Joint Perception (PJP) question gathers a single patient-reported outcome to measure how patients appraise their joint. This study compares PJP to the Forgotten Joint Score (FJS) at 21 months post-operation to assess its value. METHODS: A retrospective review was performed at an orthopedic specialty hospital for patients who completed both PJP and FJS questionnaires in 2020-2021 and underwent either a unilateral elective primary Total Knee Arthroplasty (TKA) or Total Hip Arthroplasty (THA). Spearman's correlation coefficients and P-values were calculated to determine external validity of PJP. Floor and ceiling effects were analyzed and considered present if ≥ 15% of patients achieved the worst or best score (0-4 for PJP and 0-100 for FJS). RESULTS: In total, 534 patients (327 THA and 207 TKA) were surveyed at 21 months post-operation. External validity against FJS was assessed for both TKA (r = 0.66, P < .01) and THA (r = 0.69, P < .01). For TKA, the floor and ceiling effects were 0.97% and 25.12% for PJP and 3.86% and 4.83% for FJS, respectively. For THA, the floor and ceiling effects were 0.92% and 50.46% for PJP and 2.47% and 20.50% for FJS, respectively. CONCLUSION: The PJP was correlated with FJS moderately for both TKA and THA and can be collected with lesser burden. However, ceiling effects were higher in both TKA and THA for PJP compared to FJS. Further studies are needed to investigate the questionnaires at additional time points and to evaluate the implications of high ceiling effects.