Myeloid-intrinsic cell cycle-related kinase drives immunosuppression to promote tumorigenesis.

Massive expansion of immature and suppressive myeloid cells is a common feature of malignant solid tumors. Over-expression of cyclin-dependent kinase 20, also known as cell cycle-related kinase (CCRK), in hepatocellular carcinoma (HCC) correlates with reduced patient survival and low immunotherapy responsiveness. Beyond tumor-intrinsic oncogenicity, here we demonstrated that CCRK is upregulated in myeloid cells in tumor-bearing mice and in patients with HCC. Intratumoral injection of Ccrk-knockdown myeloid-derived suppressor cells (MDSCs) increased tumor-infiltrating CD8+T cells and suppressed HCC tumorigenicity. Using an indel mutant transgenic model, we showed that Ccrk inactivation in myeloid cells conferred a mature phenotype with elevated IL-12 production, driving Th1 responses and CD8+T cell cytotoxicity to reduce orthotopic tumor growth and prolong survival. Mechanistically, CCRK activates STAT3/E4BP4 signaling in MDSCs to acquire immunosuppressive activity through transcriptional IL-10 induction and IL-12 suppression. Taken together, our findings unravel mechanistic insights into MDSC-mediated immunosuppression and offer a therapeutic kinase-target for cancer immunotherapy.

RETRACTED: Parallel bimodal single-cell sequencing of transcriptome and methylome provides molecular and translational insights on oocyte maturation and maternal aging.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. It has been brought to our attention that the authors of the article "Parallel bimodal single-cell sequencing of transcriptome and methylome provides molecular and translational insights on oocyte maturation and maternal aging" cannot agree on who should be listed as an author of the article. Further inquiry by the journal revealed that the authorship was also changed at the revision stages of the article without notifying the handling Editor, which is contrary to the journal policy on changes to authorship. The journal considers this unacceptable practice, and the Editor-in-Chief decided to retract the article.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Comparative analysis of single-cell parallel sequencing approaches in oocyte application.

Single-cell parallel sequencing allows us to explore how genetic and epigenetic variations correlate of gene expression in the same cell. Beads-based approach and non-beads-based approach are the two present methods to separate DNA and RNA from the same cell. However, systematic difference between the two methods are lacking. In our study, we compared the performances of the two methods using transcriptome and methylome profiles generated simultaneously from single mouse oocytes. Our results showed that the beads-based approach could capture maximum quantity of mRNA but loss of DNA was inevitable, while the non-beads-based approach could obtain more DNA due to the undamaged nucleus obtained but at a cost of partial loss of mRNA. As the sequencing coverage of methylome sequencing in a single cell was relatively low, single-cell whole genome bisulfite sequencing (scWGBS) was preferable to generate the methylome map in single-cell parallel sequencing in comparison to single-cell reduced representation bisulfite sequencing (scRRBS). To the best of our knowledge, this is the first study to compare the two methods of single-cell parallel sequencing which offers a basic idea for deciding between the two methods and a direction of single-cell parallel sequencing development.

An inflammatory-CCRK circuitry drives mTORC1-dependent metabolic and immunosuppressive reprogramming in obesity-associated hepatocellular carcinoma.

Obesity increases the risk of hepatocellular carcinoma (HCC) especially in men, but the molecular mechanism remains obscure. Here, we show that an androgen receptor (AR)-driven oncogene, cell cycle-related kinase (CCRK), collaborates with obesity-induced pro-inflammatory signaling to promote non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Lentivirus-mediated Ccrk ablation in liver of male mice fed with high-fat high-carbohydrate diet abrogates not only obesity-associated lipid accumulation, glucose intolerance and insulin resistance, but also HCC development. Mechanistically, CCRK fuels a feedforward loop by inducing STAT3-AR promoter co-occupancy and transcriptional up-regulation, which in turn activates mTORC1/4E-BP1/S6K/SREBP1 cascades via GSK3ß phosphorylation. Moreover, hepatic CCRK induction in transgenic mice stimulates mTORC1-dependent G-csf expression to enhance polymorphonuclear myeloid-derived suppressor cell recruitment and tumorigenicity. Finally, the STAT3-AR-CCRK-mTORC1 pathway components are concordantly over-expressed in human NASH-associated HCCs. These findings unveil the dual roles of an inflammatory-CCRK circuitry in driving metabolic and immunosuppressive reprogramming through mTORC1 activation, thereby establishing a pro-tumorigenic microenvironment for HCC development.

A Novel Calibration Procedure of Pulse Transit Time based Blood Pressure measurement with Heart Rate and Respiratory Rate.

Pulse transit time (PTT) has been a promising non-invasive and cuff-less method to measure blood pressure (BP). However, to achieve acceptable accuracy, subject specific and frequent calibration is required to model the complex non-Iinear dynamic between BP and PTT, limiting the practicality of PTT-based methods. The BP-PTT relationship is often modelled by the Moens-Korteweg (M-K) equation. Previous studies have found that the M-K equation is only able to model high frequency variations of BP, with low frequency variations modulated by the vasomotor tone, which is regulated by the sympathetic nervous system (SNS). In this paper we present a novel calibration procedure that accounts for the influence of the SNS on the BP-PTT relationship. This is achieved by identifying the state of the SNS during calibration based on heart rate (HR), respiratory rate (RR), and PTT itself. The performance of the proposed procedure has been tested on 10 subjects over a period of 4 weeks. The results showed that the measurement differences were 1.04 mmHg ± 6.88 mmHg, and -2.16mmHg ± 6.60 mmHg for systolic and diastolic blood pressure, respectively. Furthermore, the proposed procedure was found to make a significant improvement of measurement precision when compared to the previous one-point calibration method, demonstrating the potential of the new procedure for accurate long-term BP tracking.

Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) contribute to tumour immunosuppressive microenvironment and immune-checkpoint blockade resistance. Emerging evidence highlights the pivotal functions of cyclin-dependent kinases (CDKs) in tumour immunity. Here we elucidated the role of tumour-intrinsic CDK20, or cell cycle-related kinase (CCRK) on immunosuppression in hepatocellular carcinoma (HCC). DESIGN: Immunosuppression of MDSCs derived from patients with HCC and relationship with CCRK were determined by flow cytometry, expression analyses and co-culture systems. Mechanistic studies were also conducted in liver-specific CCRK-inducible transgenic (TG) mice and Hepa1-6 orthotopic HCC models using CRISPR/Cas9-mediated Ccrk depletion and liver-targeted nanoparticles for interleukin (IL) 6 trapping. Tumorigenicity and immunophenotype were assessed on single or combined antiprogrammed death-1-ligand 1 (PD-L1) therapy. RESULTS: Tumour-infiltrating CD11b+CD33+HLA-DR- MDSCs from patients with HCC potently inhibited autologous CD8+T cell proliferation. Concordant overexpression of CCRK and MDSC markers (CD11b/CD33) positively correlated with poorer survival rates. Hepatocellular CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR- MDSC expansion from human peripheral blood mononuclear cells through upregulating IL-6. Mechanistically, CCRK activated nuclear factor-κB (NF-κB) via enhancer of zeste homolog 2 (EZH2) and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. Hepatic CCRK induction in TG mice activated the EZH2/NF-κB/IL-6 cascade, leading to accumulation of polymorphonuclear (PMN) MDSCs with potent T cell suppressive activity. In contrast, inhibiting tumorous Ccrk or hepatic IL-6 increased interferon γ+tumour necrosis factor-α+CD8+ T cell infiltration and impaired tumorigenicity, which was rescued by restoring PMN-MDSCs. Notably, tumorous Ccrk depletion upregulated PD-L1 expression and increased intratumorous CD8+ T cells, thus enhancing PD-L1 blockade efficacy to eradicate HCC. CONCLUSION: Our results delineate an immunosuppressive mechanism of the hepatoma-intrinsic CCRK signalling and highlight an overexpressed kinase target whose inhibition might empower HCC immunotherapy.

ICA1L forms BAR-domain complexes with PICK1 and is crucial for acrosome formation in spermiogenesis.

Mutations in the Pick1 gene cause globozoospermia, a male infertility disorder, in both mice and humans. PICK1 is crucial for vesicle trafficking, and its deficiency in sperm cells leads to abnormal vesicle trafficking from the Golgi to the acrosome. This eventually disrupts acrosome formation and leads to male infertility. Here, we identified ICA1L, which has sequence similarities to ICA69 (also known as ICA1), as a new BAR-domain binding partner of PICK1. ICA1L is expressed in testes and brain, and is the major binding partner for PICK1 in testes. ICA1L and PICK1 are highly expressed in spermatids and trafficked together at different stages of spermiogenesis. ICA1L-knockout mice were generated by CRISPR-Cas technology. PICK1 expression was reduced by 80% in the testes of male mice lacking ICA1L. Sperm from ICA1L-knockout mice had abnormalities in the acrosome, nucleus and mitochondrial sheath formation. Both total and mobile sperm numbers were reduced, and about half of the remaining sperm had the characteristics of globozoospermia. These defects ultimately resulted in reduced fertility of male ICA1L-knockout mice, and ICA69/ICA1L-double knockout male mice were sterile.

Systems-level quantification of division timing reveals a common genetic architecture controlling asynchrony and fate asymmetry.

Coordination of cell division timing is crucial for proper cell fate specification and tissue growth. However, the differential regulation of cell division timing across or within cell types during metazoan development remains poorly understood. To elucidate the systems-level genetic architecture coordinating division timing, we performed a high-content screening for genes whose depletion produced a significant reduction in the asynchrony of division between sister cells (ADS) compared to that of wild-type during Caenorhabditis elegans embryogenesis. We quantified division timing using 3D time-lapse imaging followed by computer-aided lineage analysis. A total of 822 genes were selected for perturbation based on their conservation and known roles in development. Surprisingly, we find that cell fate determinants are not only essential for establishing fate asymmetry, but also are imperative for setting the ADS regardless of cellular context, indicating a common genetic architecture used by both cellular processes. The fate determinants demonstrate either coupled or separate regulation between the two processes. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Our quantitative dataset with cellular resolution provides a resource for future analyses of the genetic control of spatial and temporal coordination during metazoan development.

Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs.

The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.

SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS.

Common dehydration methods of cells on biomaterials for scanning electron microscopy (SEM) include air drying, hexamethyldisilazane (HMDS) or tetramethysilane (TMS) treatment and critical point drying (CPD). On the other side, freeze-drying has been widely employed in dehydrating biological samples and also in preparing porous biomaterial scaffolds but not in preparing cells on three-dimensional (3D) biomaterials for SEM examination. In this study, we compare cells on porous hydroxyapatite (HA) prepared by air drying, HMDS and freeze-drying. The effects of fixation and using phosphate buffered saline (PBS) in the fixation were also assessed on three porous calcium phosphate (CaP) materials, namely, HA, α-tricalcium phosphate (α-TCP) and ß-tricalcium phosphate (ß-TCP) samples. There is no significant difference in samples prepared by HMDS treatment and freeze-drying viewed at low magnification. Besides, it is better not to use phosphate buffer in the fixation step for CaP materials to avoid undesirable spontaneous precipitation of CaPs. On the other hand, fewer exchanges of liquids are required for freeze-drying and hence chemical fixation may not be absolutely required for samples prepared by freeze-drying. Other technical details of the preparation were also investigated and discussed. This study suggests both HMDS and freeze-drying can be employed to dehydrate cells on 3D scaffolds for SEM examination.

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation.

BACKGROUND: The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. METHODS: We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. RESULTS: Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. CONCLUSIONS: The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.