Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics.

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution MS. By combining meiotic labels annotated from the mouse genomics informatics mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder (https://github.com/sjq111/FuncProFinder), was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of 10 genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

Biomarker clustering in autosomal dominant Alzheimer's disease.

INTRODUCTION: As the number of biomarkers used to study Alzheimer's disease (AD) continues to increase, it is important to understand the utility of any given biomarker, as well as what additional information a biomarker provides when compared to others. METHODS: We used hierarchical clustering to group 19 cross-sectional biomarkers in autosomal dominant AD. Feature selection identified biomarkers that were the strongest predictors of mutation status and estimated years from symptom onset (EYO). Biomarkers identified included clinical assessments, neuroimaging, cerebrospinal fluid amyloid, and tau, and emerging biomarkers of neuronal integrity and inflammation. RESULTS: Three primary clusters were identified: neurodegeneration, amyloid/tau, and emerging biomarkers. Feature selection identified amyloid and tau measures as the primary predictors of mutation status and EYO. Emerging biomarkers of neuronal integrity and inflammation were relatively weak predictors. DISCUSSION: These results provide novel insight into our understanding of the relationships among biomarkers and the staging of biomarkers based on disease progression.

Dynamic Responses of Chromosome-Binding Protein Complexes to Meiotic Prophase I of Mouse Spermatocyte.

Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their functions in MPI have not yet been well scrutinized. Quantitative proteomics focused on MPI-related CBPs was accomplished, in which mouse primary spermatocytes in four different subphases of MPI were collected, and chromosome-enriched proteins were extracted and quantitatively identified. According to a stringent criterion, 1136 CBPs in the MPI subphases were quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided into two groups: high abundance in leptotene and zygotene or that in pachytene and diplotene. Furthermore, 152 such CBPs were regarded as 26 CBP complexes with strict filtration, in which some of these complexes were perceived to be MPI-dependent for the first time. These complexes basically belonged to four functional categories, while their dynamic abundance changes following MPI appeared; the functions of DNA replication decreased; and transcription and synapsis were activated in zygotene, pachytene, and diplotene; in contrast to the traditional prediction, condensin activity weakened in pachytene and diplotene. Profiling of protein complexes thus offered convincing evidence of the importance of CBP complexes in MPI.

Glucose-induced, duration-dependent genome-wide DNA methylation changes in human endothelial cells.

DNA methylation, a critical epigenetic mechanism, plays an important role in governing gene expressions during biological processes such as aging, which is well known to be accelerated in hyperglycemia (diabetes). In the present study, we investigated the effects of glucose on whole genome DNA methylation in small [human retinal microvascular endothelial cells (HRECs)] and large [human umbilical vein endothelial cells (HUVECs)] vessel endothelial cell (EC) lines exposed to basal or high glucose-containing media for variable lengths of time. Using the Infinium EPIC array, we obtained 773,133 CpG sites (probes) for analysis. Unsupervised clustering of the top 5% probes identified four distinct clusters within EC groups, with significant methylation differences attributed to EC types and the duration of cell culture rather than glucose stimuli alone. When comparing the ECs incubated for 2 days versus 7 days, hierarchical clustering analyses [methylation change >10% and false discovery rate (FDR) <0.05] identified 17,354 and 128 differentially methylated CpGs for HUVECs and HRECs, respectively. Predominant DNA hypermethylation was associated with the length of culture and was enriched for gene enhancer elements and regions surrounding CpG shores and shelves. We identified 88 differentially methylated regions (DMRs) for HUVECs and 8 DMRs for HRECs (all FDR <0.05). Pathway enrichment analyses of DMRs highlighted involvement of regulators of embryonic development (i.e., HOX genes) and cellular differentiation [transforming growth factor-ß (TGF-ß) family members]. Collectively, our findings suggest that DNA methylation is a complex process that involves tightly coordinated, cell-specific mechanisms. Such changes in methylation overlap genes critical for cellular differentiation and embryonic development.

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8(+/Y) ) and ph8(-/Y) ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.

Analysis of Surfing Injuries Presenting in the Acute Trauma Setting.

BACKGROUND AND PURPOSE: Surfing is a rapidly growing major worldwide sport; however, little is understood regarding severe injuries and resulting hospital admissions. This study explores surfing-related injuries in the major surfing hub of San Diego presenting in the acute trauma setting. The purpose of this study is to address the void of information regarding severe surfing injuries in the trauma setting, including injury patterns, associated hospitalization course, and risk factors. Understanding the injury patterns in surfing accidents is crucial for proper management of surfing injuries. METHODS: A retrospective analysis was performed of all surfing-related injuries in a Level 1 trauma center between 2000 and 2016. RESULTS: A total of 93 patients were identified. Body parts most commonly affected include the head (42, 46%), face (21, 22%), and spine (47, 51%). Twenty-eight (30%) patients required surgical intervention, including 19 for spinal injuries, 3 for facial injuries, 4 for upper extremity injuries, and 2 for lower extremity injuries. The distribution for most presentations (55, 59%) occurred in the summer months between July and September. The Injury Severity Score demonstrated strong positive correlation with the length of hospital stay, with a Pearson coefficient of 0.52 (P < 0.01). The average length of hospitalization was 5.8 days, with intensive care unit level care required in 49% (46) patients and average length of intensive care unit stay of 5.5 days. Alcohol content was tested in 84% (78) of patients and found positive in 10% (8) of tested patients. Drug screening was performed in 70% (64) patients and found positive in 38% (24) of tested patients. CONCLUSIONS: Surfing, although a relatively safe sport, is not without major risks. In contrast with other studies, we found a high proportion of head, face, and spine injuries in patients presenting with surfing injuries in the trauma setting, consistent with its presentation as a high velocity and high impact injury. With plastic surgeons often treating severe head and facial injuries, understanding the injury patterns in severe surfing accidents is crucial for proper management. High rates of positive alcohol and drug screening signal the importance to bring awareness to the dangers of surfing under the influence.

Yeast Hmt1 catalyses asymmetric dimethylation of histone H3 arginine 2 in vitro.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate protein arginine residues. PRMTs' substrates include histones and a variety of non-histone proteins. Previous studies have shown that yeast Hmt1 is a type I PRMT and methylates histone H4 arginine 3 and several mRNA-binding proteins. Hmt1 forms dimers or oligomers, but how dimerization or oligomerization affects its activity remains largely unknown. We now report that Hmt1 can methylate histone H3 arginine 2 (H3R2) in vitro. The dimerization but not hexamerization is essential for Hmt1's activity. Interestingly, the methyltransferase activity of Hmt1 on histone H3R2 requires reciprocal contributions from two Hmt1 molecules. Our results suggest an intermolecular trans-complementary mechanism by which Hmt1 dimer methylates its substrates.

Expression profiles of histone lysine demethylases during cardiomyocyte differentiation of mouse embryonic stem cells.

AIM: Histone lysine demethylases (KDMs) control the lineage commitments of stem cells. However, the KDMs involved in the determination of the cardiomyogenic lineage are not fully defined. The aim of this study was to investigate the expression profiles of KDMs during the cardiac differentiation of mouse embryonic stem cells (mESCs). METHODS: An in vitro cardiac differentiation system of mESCs with Brachyury (a mesodermal specific marker) and Flk-1(+)/Cxcr4(+) (dual cell surface biomarkers) selection was used. The expression profiles of KDMs during differentiation were analyzed using Q-PCR. To understand the contributions of KDMs to cardiomyogenesis, the mESCs on differentiation d 3.5 were sorted by FACS into Brachyury(+) cells and Brachyury(-) cells, and mESCs on d 5.5 were sorted into Flk-1(+)/Cxcr4(+) and Flk-1(-)/Cxcr4(-) cells. RESULTS: mESCs were differentiated into spontaneously beating cardiomyocytes that were visible in embryoid bodies (EBs) on d 7. On d 12-14, all EBs developed spontaneously beating cardiomyocytes. Among the 16 KDMs examined, the expression levels of Phf8, Jarid1a, Jhdm1d, Utx, and Jmjd3 were increased by nearly 2-6-fold on d 14 compared with those on d 0. Brachyury(+) cells showed higher expression levels of Jmjd3, Jmjd2a and Jhdm1d than Brachyury(-) cells. A higher level of Jmjd3 was detected in Flk-1(+)/Cxcr4(+) cells, whereas the level of Jmjd2c was lower in both Brachyury(+) cells and Flk-1(+)/Cxcr4(+) cells. CONCLUSION: KDMs may play important roles during cardiomyogenesis of mESCs. Our results provide a clue for further exploring the roles of KDMs in the cardiac lineage commitment of mESCs and the potential interference of cardiomyogenesis.

ZFP541 and KCTD19 regulate chromatin organization and transcription programs for male meiotic progression.

The successful progression of meiosis prophase I requires integrating information from the structural and molecular levels. In this study, we show that ZFP541 and KCTD19 work in the same genetic pathway to regulate the progression of male meiosis and thus fertility. The Zfp541 and/or Kctd19 knockout male mice show various structural and recombination defects including detached chromosome ends, aberrant localization of chromosome axis components and recombination proteins, and globally altered histone modifications. Further analyses on RNA-seq, ChIP-seq, and ATAC-seq data provide molecular evidence for the above defects and reveal that ZFP541/KCTD19 activates the expression of many genes by repressing several major transcription repressors. More importantly, we reveal an unexpected role of ZFP541/KCTD19 in directly modulating chromatin organization. These results suggest that ZFP541/KCTD19 simultaneously regulates the transcription cascade and chromatin organization to ensure the coordinated progression of multiple events at chromosome structural and biochemical levels during meiosis prophase I.

Exploring the value of structured narrative feedback within the Serious Illness Conversation-Evaluation Exercise (SIC-Ex): a qualitative analysis.

OBJECTIVES: The Serious Illness Conversation Guide (SICG) has emerged as a framework for conversations with patients with a serious illness diagnosis. This study reports on narratives generated from open-ended questions of a novel assessment tool, the Serious Illness Conversation-Evaluation Exercise (SIC-Ex), to assess resident-led conversations with patients in oncology outpatient clinics. DESIGN: Qualitative study using template analysis. SETTING: Three academic cancer centres in Canada. PARTICIPANTS: 7 resident physicians (trainees), 7 patients from outpatient cancer clinics, 10 preceptors (raters) consisting of medical oncologists, palliative care physicians and radiation oncologists. INTERVENTIONS: Each trainee conducted an SIC with a patient, which was videotaped. The raters watched the videos and evaluated each trainee using the novel SIC-Ex and the reference Calgary-Cambridge Guide (CCG) initially and again 3 months later. Two independent coders used template analysis to code the raters' narrative comments and identify themes/subthemes. OUTCOME MEASURES: How narrative comments aligned with elements of the CCG and SICG. RESULTS: Template analysis yielded four themes: adhering to SICG, engaging patients and family members, conversation management and being mindful of demeanour. Narrative comments identified numerous verbal and non-verbal elements essential to SICG. Some comments addressing general skills in engaging patients/families and managing the conversation (eg, setting agenda, introduction, planning, exploring, non-verbal communication) related to both the CCG and SICG, whereas other comments such as identifying substitute decision maker(s), affirming commitment and introducing Advance Care Planning were specific to the SICG. CONCLUSIONS: Narrative comments generated by SIC-Ex provided detailed and nuanced insights into trainees' competence in SIC, beyond the numerical ratings of SIC-Ex and the general communication skills outlined in the CCG, and may contribute to a more fulsome assessment of SIC skills.

Desipramine reverses remote memory deficits by activating calmodulin-CaMKII pathway in a UTX knockout mouse model of Kabuki syndrome.

Background: Kabuki syndrome (KS) is a rare developmental disorder characterised by multiple congenital anomalies and intellectual disability. UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), which encodes a histone demethylase, is one of the two major pathogenic risk genes for KS. Although intellectual disability is a key phenotype of KS, the role of UTX in cognitive function remains unclear. Currently, no targeted therapies are available for KS. Aims: This study aimed to investigate how UTX regulates cognition, to explore the mechanisms underlying UTX dysfunction and to identify potential molecular targets for treatment. Methods: We generated UTX conditional knockout mice and found that UTX deletion downregulated calmodulin transcription by disrupting H3K27me3 (trimethylated histone H3 at lysine 27) demethylation. Results: UTX-knockout mice showed decreased phosphorylation of calcium / calmodulin-dependent protein kinase II, impaired long-term potentiation and deficit in remote contextual fear memory. These effects were reversed by an Food and Drug Administration-approved drug desipramine. Conclusions: Our results reveal an epigenetic mechanism underlying the important role of UTX in synaptic plasticity and cognitive function, and suggest that desipramine could be a potential treatment for KS.

The hydroxylation activity of Jmjd6 is required for its homo-oligomerization.

Jumonji C-terminal (JmjC) domain-containing proteins are protein hydroxylases and histone demethylases that control gene expression. Jumonji domain-containing protein 6 (Jmjd6) is indispensable for embryonic development and has both histone arginine demethylase and lysyl-hydroxylase activities. The protein undergoes post-translational homo-oligomerization, but the underlying mechanism remains unknown. In this study, we examined the enzymatic activity of Jmjd6 and uncovered the mechanism underlying its homo-oligomerization. An in vitro enzymatic assay monitored by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry indicates that Jmjd6 is unable to remove the methyl group from histone arginine residues but can hydroxylate the histone H4 tail at lysine residues in a 2-oxoglutarate (2-OG)- and Fe (II)-dependent manner. A mutational analysis reveals that the homo-oligomerization of Jmjd6 requires its enzymatic activity and the N- and C-termini. Using an in vitro enzymatic assay, we further demonstrate that Jmjd6 can hydroxylate its N-terminus but not its C-terminus. In summary, we did not detect arginine demethylase activity for Jmjd6, but we did confirm that it could catalyze the lysyl-hydroxylation of histone peptides. In addition, we demonstrated that the homo-oligomerization of Jmjd6 requires its own enzymatic activity and the N- and C-termini. We propose that Jmjd6 forms intermolecular covalent bonds between its N- and C-termini via autohydroxylation.

Direct thrombolytic therapy in portal and mesenteric vein thrombosis.

A 34-year-old female from Laos presented to the emergency department with a 3-week history of worsening abdominal pain; she subsequently developed an acute abdomen requiring emergent exploratory laparotomy. An intraoperative angiography was performed, which revealed complete portal vein thrombosis. A 5F 20-cm infusion catheter was placed through an omental vein, and tissue plasminogen activator was administered directly into the catheter with successful decrease in thrombus burden. There are few controlled data on which to base clinical decisions in patients with portal vein thrombosis. Our case expands on these earlier reports that direct thrombolysis can be safely performed using local, intraclot infusions for portal vein thrombosis, and thrombolytic doses can be kept relatively low, limiting the risk of bleeding complications.

Disrupting PHF8-TOPBP1 connection elicits a breast tumor-specific vulnerability to chemotherapeutics.

The DNA damage response (DDR) pathway generally protects against genome instability, and defects in DDR have been exploited therapeutically in cancer treatment. We have reported that histone demethylase PHF8 demethylates TOPBP1 K118 mono-methylation (K118me1) to drive the activation of ATR kinase, one of the master regulators of replication stress. However, whether dysregulation of this physiological signalling is involved in tumorigenesis remains unknown. Here, we showed PHF8-promoted TOPBP1 demethylation is clinically associated with breast tumorigenesis and patient survival. Mammary gland tumors from Phf8 knockout mice grow slowly and exhibit higher level of K118me1, lower ATR activity, and increased chromosomal instability. Importantly, we found that disruption of PHF8-TOPBP1 axis suppresses breast tumorigenesis and creates a breast tumor-specific vulnerability to PARP inhibitor (PARPi) and platinum drug. CRISPR/Cas9 mutation modelling of the deleted or truncated mutation of PHF8 in clinical tumor samples demonstrated breast tumor cells expressing the mimetic variants are more vulnerable to PARPi. Together, our study supports the pursuit of PHF8-TOPBP1 signalling pathway as promising avenues for targeted therapies of PHF8-TOPBP1 proficient tumors, and provides proof-of-concept evidence for loss-of-function of PHF8 as a therapeutic indicator of PARPis.

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαß+CD8αα+ intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαß+CD8αα+ IELs. In the absence of Kdm6b, TCRαß+CD8αα+ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαß+CD8αα+ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαß+CD8αα+ IELs (IELPs) to IL-15 and TGF-ß. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαß+CD8αα+ IELs.

Molecular determinants of resistance to antiandrogen therapy.

Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.