Parallel bimodal single-cell sequencing of transcriptome and chromatin accessibility.

Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)-centered upstream regulatory networks for various cell states.

Characterization, reactivity, source apportionment, and potential source areas of ambient volatile organic compounds in a typical tropical city.

Based on one-year observation, the concentration, sources, and potential source areas of volatile organic compounds (VOCs) were comprehensively analyzed to investigate the pollution characteristics of ambient VOCs in Haikou, China. The results showed that the annual average concentration of total VOCs (TVOCs) was 11.4 ppbV, and the composition was dominated by alkanes (8.2 ppbV, 71.4%) and alkenes (1.3 ppbV, 20.5%). The diurnal variation in the concentration of dominant VOC species showed a distinct bimodal distribution with peaks in the morning and evening. The greatest contribution to ozone formation potential (OFP) was made by alkenes (51.6%), followed by alkanes (27.2%). The concentrations of VOCs and nitrogen dioxide (NO2) in spring and summer were low, and it was difficult to generate high ozone (O3) concentrations through photochemical reactions. The significant increase in O3 concentrations in autumn and winter was mainly related to the transmission of pollutants from the northeast. Traffic sources (40.1%), industrial sources (19.4%), combustion sources (18.6%), solvent usage sources (15.5%) and plant sources (6.4%) were identified as major sources of VOCs through the positive matrix factorization (PMF) model. The southeastern coastal areas of China were identified as major potential source areas of VOCs through the potential source contribution function (PSCF) and concentration-weighted trajectory (CWT) models. Overall, the concentration of ambient VOCs in Haikou was strongly influenced by traffic sources and long-distance transport, and the control of VOCs emitted from vehicles should be strengthened to reduce the active species of ambient VOCs in Haikou, thereby reducing the generation of O3.

[Primary urothelial carcinoma of the prostate: A case report and review of the literature].

OBJECTIVE: To investigate the clinical diagnosis, treatment and prognosis of primary urothelial carcinoma of the prostate. METHODS: We retrospectively analyzed the clinical data on one case of primary urothelial carcinoma of the prostate and reviewed the relevant published literature. RESULTS: The patient, aged 83 years old and diagnosed with primary urothelial carcinoma of the prostate, was treated by neoadjuvant chemotherapy with gemcitabine plus cisplatin preoperatively, injected with 5-fluorouracil into the cutting edge of the tumor intraoperatively, and pathologically confirmed with high-grade invasive primary urothelial carcinoma of the prostate, followed by adjuvant chemotherapy with gemcitabine plus cisplatin. No recurrence or metastasis was observed during 1-year follow-up. CONCLUSIONS: Primary urothelial carcinoma of the prostate is rare and highly malignant. If the tumor is localized, the patient needs to be treated by neoadjuvant chemotherapy preoperatively, injection with anti-tumor drugs into the cutting edge of the tumor intraoperatively, and adjuvant chemotherapy and regular follow-up postoperatively.

[Detection and Analysis of Human Parainfluenza Virus Infection in Hospitalized Adults with Acute Respiratory Tract Infections].

OBJECTIVE: To investigate the prevalence and gene characteristics of different groups of human parainfluenza virus (HPIV) infection in hospitalized adults with acute respiratory tract infections (ARI). METHODS: RT-PCR was used to detect HPIV hemagglutinin (HA) DNA,which was extracted from sputum samples of 1 039 adult patients with ARI from March,2014 to June,2016. The HA gene amplified from randomly selected positive samples were sequenced to analyze the homology and variation. RESULTS: 10.6% (110/1 039) of these samples were positive for HPIV,including 8 cases of HPIV-1,22 cases of HPIV-2,46 cases of HPIV-3 and 34 cases of HPIV-4. Detectable rate varied among different groups of HPIV according to seasons of the year and ages of patients. No significant differences were found between the positive samples and the reference sequences. Compared with different reference strains of different regions,the genetic distance of nucleotide is the smallest between the strains tested in this study and the reference strains of other provinces and cities in China. CONCLUSION: In Chengdu region,HPIV virus is highly detected in ARI,all subtypes were detected with HPIV-3 being the main subtype.

Hyper-IL-15 suppresses metastatic and autochthonous liver cancer by promoting tumour-specific CD8+ T cell responses.

BACKGROUND & AIMS: Liver cancer has a very dismal prognosis due to lack of effective therapy. Here, we studied the therapeutic effects of hyper-interleukin15 (hyper-IL-15), which is composed of IL-15 and the sushi domain of the IL-15 receptor α chain, on metastatic and autochthonous liver cancers. METHODS: Liver metastatic tumour models were established by intraportally injecting syngeneic mice with murine CT26 colon carcinoma cells or B16-OVA melanoma cells. Primary hepatocellular carcinoma (HCC) was induced by diethylnitrosamine (DEN). A hydrodynamics-based gene delivery method was used to achieve sustained hyper-IL-15 expression in the liver. RESULTS: Liver gene delivery of hyper-IL-15 robustly expanded CD8(+) T and NK cells, leading to a long-term (more than 40 days) accumulation of CD8(+) T cells in vivo, especially in the liver. Hyper-IL-15 treatment exerted remarkable therapeutic effects on well-established liver metastatic tumours and even on DEN-induced autochthonous HCC, and these effects were abolished by depletion of CD8(+) T cells but not NK cells. Hyper-IL-15 triggered IL-12 and interferon-γ production and reduced the expression of co-inhibitory molecules on dendritic cells in the liver. Adoptive transfer of T cell receptor (TCR) transgenic OT-1 cells showed that hyper-IL-15 preferentially expanded tumour-specific CD8(+) T cells and promoted their interferon-γ synthesis and cytotoxicity. CONCLUSIONS: Liver delivery of hyper-IL-15 provides an effective therapy against well-established metastatic and autochthonous liver cancers in mouse models by preferentially expanding tumour-specific CD8(+) T cells and promoting their anti-tumour effects.

[Functional outcomes of transvesical single-site versus extraperitoneal laparoscopic radical prostatectomy for low-risk prostate cancer].

OBJECTIVE: To compare the perioperative data, pathological results and functional outcomes of transvesical single- site laparoscopic radical prostatectomy (TVSSLRP) with those of nerve-sparing extraperitoneal laparoscopic radical prostatectomy (nsELRP) in the treatment of low-risk prostate cancer (PCa). METHODS: Fifty patients with low-risk organ-confined PCa were randomly assigned to two groups of equal number to receive TVSSLRP and nsELRP, respectively. Comparisons were made between the two groups of patients in such demographic and baseline data as age, comorbidity, body mass index (BMI), serum prostate-specific antigen (PSA), prostate volume, bioptic Gleason score, clinical stage, IIEF-5 score, nocturnal penile tumescence (NPT), penile brachial index (PBI), and penile arterial blood flow velocity as well as in such surgery-related parameters as operation duration, blood loss, blood transfusion, intraoperative complications, positive surgical margin, catheterization time, hospital stay, and postoperative Gleason score, pathologic stage, urinal pad use, PSA level, IIEF-5 score, NPT, PBI and PABFV. RESULTS: All the operations were successfully performed. There were no statistically significant differences between the two groups either in the demographic and baseline data or in intraoperative blood loss, blood transfusion rate, complications, and positive surgical margin. No intraoperative complications and positive surgical margins were found in either group. Compared with nsELRP, TVSSLRP achieved a significantly shorter operation duration ([151.46 ± 40.68] min vs [105.92 ± 26.21] min, P <0.05), catheterization time ([13.01 ± 1.64] d vs [11.24 ± 1.17] d, P <0.05), and hospital stay ([15.76 ± 4.65] d vs [12.92 ± 4.29] d, P <0.05). On the first day and at 1, 3 and 6 months after catheter removal, the urinary continence rates in the TVSSLRP and nsELRP groups were 84% vs 52% (P <0.05), 100% vs 84%, 100% vs 96%, and 100% vs 96%, respectively; and at 3, 6 and 12 months after surgery, the erectile potency rates were 48% vs 28% (P <0.05), 64% vs 52%, and 76% vs 68%, respectively, with an IIEF-5 score ≥ 18, all evidently higher in the TVSSLRP than in the nsELRP group. The penile brachial index and arterial blood flow velocity of the two groups of patients exhibited no significant differences before and after surgery, nor did postoperative complications (grade II) between the TVSSLRP and nsELRP groups (32% vs 40%, P >0.05). The Gleason score and pathologic stage were increased after surgery, but with remarkable differences between the two groups (P >0.05). No biochemical recurrence was found in either group during a 12-month follow-up. CONCLUSION: With the advantages of safety and rapid postoperative recovery, both TVSSLRP and nsELRP are feasible for the treatment of low-risk organ-confined PCa, but the former may achieve an earlier recovery of urinary continence and erectile function than the latter.

Characteristics and ecological risks of microplastic pollution in a tropical drinking water source reservoir in Hainan province, China.

Microplastic (<5 mm) pollution has become a pressing environmental concern in recent years. The present study investigated the occurrence characteristics and assessed the ecological risk of microplastics in the surface water and sediment of the Chitian Reservoir, a drinking water source in Hainan province (China). The results indicated that microplastics were detected in the surface water and sediment of the Chitian Reservoir and its surrounding areas. The overall abundance of microplastics in the water was 3.05 ± 1.16 items per L and in the sediment was 0.15 ± 0.06 items per g dry weight, which is relatively low compared to other reservoirs in China. The dominant components of microplastics detected in the Chitian Reservoir were polypropylene (PP), rayon, and polyester. Physical morphology analysis of microplastics showed that fibers with small particle sizes (<1 mm) and white color were the predominant characteristics in both the surface water and sediment. The domestic sewage from surrounding residents and agricultural wastewater may be the primary sources of microplastics in the reservoir. Ecological risk assessment revealed that the overall pollution load index (PLI) in the surface water (0.65) and sediment (0.51) of the Chitian Reservoir and its surrounding area is at a low level. The potential ecological hazards (RI) of microplastics (0.13 to 336.78 in water; 0.23 to 465.93 in sediment) in most sites fall within the scope of level I, but those in a few sites are at level II due to the presence of polyvinyl chloride (PVC). This study enriches the data on microplastic pollution in inland reservoir systems, providing fundamental reference information for future ecotoxicological studies and the management of microplastic pollution control.

P-doped spherical hard carbon with high initial coulombic efficiency and enhanced capacity for sodium ion batteries.

Hard carbon (HC) is one of the most promising anode materials for sodium-ion batteries (SIBs) due to its cost-effectiveness and low-voltage plateau capacity. Heteroatom doping is considered as an effective strategy to improve the sodium storage capacity of HC. However, most of the previous heteroatom doping strategies are performed at a relatively low temperature, which could not be utilized to raise the low-voltage plateau capacity. Moreover, extra doping of heteroatoms could create new defects, leading to a low initial coulombic efficiency (ICE). Herein, we propose a repair strategy based on doping a trace amount of P to achieve a high capacity along with a high ICE. By employing the cross-linked interaction between glucose and phytic acid to achieve the in situ P doped spherical hard carbon, the obtained PHC-0.2 possesses a large interlayer space that facilitates Na+ storage and transportation. In addition, doping a suitable amount of P could repair some defects in carbon layers. When used as an anode material for SIBs, the PHC-0.2 exhibits an enhanced reversible capacity of 343 mA h g-1 at 20 mA g-1 with a high ICE of 92%. Full cells consisting of a PHC-0.2 anode and a Na2Fe0.5Mn0.5[Fe(CN)6] cathode exhibited an average potential of 3.1 V with an initial discharge capacity of 255 mA h g-1 and an ICE of 85%. The full cell displays excellent cycling stability with a capacity retention of 80.3% after 170 cycles. This method is simple and low-cost, which can be extended to other energy storage materials.

Loss of epithelial oestrogen receptor α inhibits oestrogen-stimulated prostate proliferation and squamous metaplasia via in vivo tissue selective knockout models.

Squamous metaplasia (SQM) is a specific phenotype in response to oestrogen in the prostate and oestrogen receptor (ER) α is required to mediate this response. Previous studies utilizing tissue recombination with seminal vesicle (SV) mesenchyme and prostatic ductal tips from wild type and ERαKO mice suggested that both epithelial and stromal ERα are necessary for SQM. However, tissue recombination is conducted in the renal capsule of immune-deficient mice, in which the microenvironment is different from normal prostate microenvironment in the intact mice. Furthermore, whether the requirement of stromal ERα in the SV for developing SQM is the same as in the prostate is unknown. Therefore, there is a clear need to evaluate the respective roles of ERα in prostate epithelial versus stromal compartments in the intact mouse. Here we generated a mouse model that has selectively lost ERα in either stromal (FSP-ERαKO) or epithelial prostate cells (pes-ERαKO) to determine the requirements of ERα for oestrogen-stimulated prostate proliferation and SQM. Our results indicated that FSP-ERαKO prostates develop full and uniform SQM, which suggests that loss of the majority (~65%) of stromal ERα will not influence oestrogen-mediated SQM. In contrast, loss of epithelial ERα inhibits oestrogen-mediated prostate growth and SQM evidenced by decreasing cytokertin 10 positive squamous cell stratification and differentiation, by reduced ERα protein expression in SQM compared to wild type mice ERα, and by the presence of normal proliferative activities in the oestrogen-treated pes-ERαKO prostates. These in vivo results suggest that epithelial ERα is required for oestrogen-mediated proliferative response and could be an appropriate target for preventing aberrant oestrogen signalling in the prostate.

Occurrence and ecological risks of flonicamid and its metabolites in multiple substrates from intensive rice-vegetable rotations in tropical China.

Rice-vegetable rotations are dominant in (sub)-tropical agriculture worldwide. However, fate and risks of the insecticide flonicamid (FLO) and its main degradates (collectively called FLOMs) in multiple substrates from those cropping systems remain largely unknown. In this study, we characterized residual concentrations, driving factors, transport, and potential ecological risks of FLOMs in different substrates in 28 tropical rice-vegetable rotations. Concentrations (median) of FLOMs were 0.013-3.03 (0.42) ng g-1 in plants, 0.012-1.92 (0.23) ng g-1 in soil, 0.029-0.63 (0.126) µg L-1 in water, and 0.002-0.398 (0.055) ng g-1 in sediments. Flonicamid and its metabolite N-(4-trifluoromethylnicotinoyl) glycine were the dominant species in the four substrates (84.1 % to 88.5 %). Plants had the highest levels of FLOMs, with the highest bioconcentration factor in peppers. According to boosted regression trees coupled with a partial least squares structural equation model, levels and composition of FLOMs showed high spatiotemporal and crop-related patterns in different substrates, with patterns highly codetermined by agricultural practices (e.g., crop type and FLO/neonicotinoid/pyrethroid applications), substrate parameters (e.g., pH, organic matter or total organic carbon), and climate features (e.g., wet/dry seasons). Moreover, a fugacity method indicated differences in transport and partitioning patterns in different substrates during rice and vegetable planting periods. Integrated substrate risk assessment of FLOMs contamination was conducted based on species-sensitive distributions and substrate weight index. Although overall risks of FLOM contamination in tropical rice-vegetable rotations were negligible to low, the highest risks were in soils, vegetable planting periods, and a central intensively planted area.

[Ozone Pollution Characteristics and Formation Mechanism in a Typical Tropical Seaside City].

To investigate the pollution characteristics and formation mechanism of ambient air ozone(O3) in a typical tropical seaside city, we conducted an observational experiment on O3 and its precursors at an urban site in Haikou, Hainan Province, from June to October 2019. The O3 pollution characteristics were analyzed comprehensively; the O3-NOx-VOCs sensitivities and key precursors were determined, and the control strategies for O3 pollution were carried out. The results were as follows:1 O3 pollution in Haikou mainly occurred in September and October, with daily maximum 8-h O3 concentrations in the range of 39-190 µg·m-3, and the daily variation in O3 was unimodal, peaking at approximately 14:00. 2 The concentrations of NO2 and VOCs were higher during O3 pollution episodes than their respective mean values in Haikou City. The increased O3 precursor concentrations were an important factor leading to O3 pollution, whereas O3 pollution was also influenced by regional transport, with pollutants mainly transported from the northeastern part of Haikou City. 3 O3-NOx-VOCs sensitivity in Haikou City was in the VOCs and NOx transitional regime, and the most sensitive precursors in various months were different. O3 formation in September was sensitive to anthropogenic VOCs the most; however, in October it was sensitive to NOx. 4 In the future, the reduction ratio of VOCs to NOx should be 1:1-4:1 to control O3 pollution effectively in Haikou.

Effects of a combination of oral anti-diabetes drugs with basal insulin therapy on ß-cell function and glycaemic control in patients with newly diagnosed type 2 diabetes.

BACKGROUND: Oral anti-diabetes drugs plus basal insulin (OAD + insulin) therapy in patients with newly diagnosed type 2 diabetes might improve ß-cell function and result in extended glycaemic remission. This randomised trial compared the effect on ß-cell function and diabetes remission rate between oral drug alone or with addition of basal insulin. METHODS: One hundred and twenty-nine patients, aged 35-50 years, were enrolled between June 2005 and June 2009. For initial correction of hyperglycaemia, patients with fasting plasma glucose ≥9.0 mmol/L and HbA(1c) ≥ 9.0%, were randomly assigned to therapy with oral drugs + insulin or oral drugs alone. Treatment was stopped after normoglycaemia was maintained for 3 months. Patients were then followed-up with diet and physical exercise. Blood glucose, HbA(1c) and insulin were measured prior to treatment and at 1-year follow-up. RESULTS: More patients achieved target glycaemic control in the oral drugs + insulin group [98.3% (58 of 59)] in less time [(10.4 ± 2.5) days] than those in the oral drug group [95.7% (67 of 70) and (12.4 ± 3.4) days]. At 1-year follow-up, more patients maintained target glycaemia without any drugs in the oral drug + insulin group than in the oral drug group [37.9% (22 of 58) vs 20.9% (14 of 67)]. Both treatments improved homeostasis model assessment-ß (HOMA-ß) and homeostasis model assessment-insulin resistance (HOMA-IR) significantly. They had similar effects on insulin resistance [lg(HOMA-IR): (0.50 ± 0.09) vs (0.48 ± 0.09), p = 0.23]. However, oral drugs + insulin could recover ß-cell function much more than OAD alone could [lg(HOMA-ß): (2.17 ± 0.14) vs (2.11 ± 0.13), p = 0.03]. CONCLUSION: In newly diagnosed type 2 diabetes, therapy with oral drugs + insulin has had favourable outcomes on recovery and maintenance of ß-cell function and protracted glycaemic remission compared with treatment with oral drugs alone.

68Ga-PSMA ligand PET/CT integrating indocyanine green-guided salvage lymph node dissection for lymph node metastasis after radical prostatectomy.

To efficiently remove all recurrent lymph nodes (rLNs) and minimize complications, we developed a combination approach that consisted of 68Gallium prostate-specific membrane antigen (PSMA) ligand positron emission tomography (PET)/computed tomography (CT) and integrated indocyanine green (ICG)-guided salvage lymph node dissection (sLND) for rLNs after radical prostatectomy (RP). Nineteen patients were enrolled to receive such treatment. 68Ga-PSMA ligand PET/CT was used to identify rLNs, and 5 mg of ICG was injected into the space between the rectum and bladder before surgery. Fluorescent laparoscopy was used to perform sLND. While extensive LN dissection was performed at level I, another 5 mg of ICG was injected via the intravenous route to intensify the fluorescent signal, and laparoscopy was introduced to intensively target stained LNs along levels I and II, specifically around suspicious LNs, with 68Ga-PSMA ligand PET/CT. Next, both lateral peritonea were exposed longitudinally to facilitate the removal of fluorescently stained LNs at levels III and IV. In total, pathological analysis confirmed that 42 nodes were rLNs. Among 145 positive LNs stained with ICG, 24 suspicious LNs identified with 68Ga-PSMA ligand PET/CT were included. The sensitivity and specificity of 68Ga-PSMA ligand PET/CT for detecting rLNs were 42.9% and 96.6%, respectively. For ICG, the sensitivity was 92.8% and the specificity was 39.1%. At a median follow-up of 15 (interquartile range [IQR]: 6-31) months, 15 patients experienced complete biochemical remission (BR, prostate-specific antigen [PSA] <0.2 ng ml-1), and 4 patients had a decline in the PSA level, but it remained >0.2 ng ml-1. Therefore, 68Ga-PSMA ligand PET/CT integrating ICG-guided sLND provides efficient sLND with few complications for patients with rLNs after RP.

Sildenafil improves right ventricular remodelling in monocrotaline-induced rats by decreasing myocardial apoptosis and activating peroxisome proliferator-activated receptors.

OBJECTIVES: To assess the effect of sildenafil on monocrotaline-induced right ventricular (RV) remodeling and investigate the possible mechanism. METHODS: Rats were subcutaneously injected with monocrotaline to establish an RV remodeling model and then administered sildenafil (25 mg/kg) from days 1 to 28. After 28 days of administration, the RV systolic pressure and the RV hypertrophy index (RVHI) were measured. The morphology of the right ventricle was observed by H&E staining. The ultrastructure of the right ventricle was observed using a transmission electron microscope. The myocardial apoptosis of the right ventricle was evaluated by TUNEL staining. The protein expression of apoptosis-related proteins and PPARs were examined by western blotting. KEY FINDINGS: The results indicated that sildenafil decreased the RV systolic pressure and RVHI, and improved the microstructure and ultrastructure of the right ventricle in monocrotaline-induced rats. In addition, sildenafil suppressed myocardial apoptosis and promoted the protein expression of PPARs of the right ventricle in monocrotaline-induced rats. CONCLUSION: Sildenafil inhibits RV remodeling in monocrotaline-induced rats, which might be partially mediated by reducing myocardial apoptosis and activating PPARs.

Voltage-assisted SILAR deposition of CdSe quantum dots to construct a high performance of ZnS/CdSe/ZnS quantum dot-sensitized solar cells.

The charge recombination on the interfaces of TiO2/quantum dots (QDs)/electrolyte is a key factor limiting the efficiency of quantum dot-sensitized solar cells (QDSSCs). Construction of double-layer barrier structure of ZnS/QDs/ZnS is a vital strategy to suppress the interfacial charge recombination. However, a large lattice mismatch (12%) at CdSe/ZnS interfaces causes CdSe to grow slowly on TiO2/ZnS mesoporous film, weakening the interaction between QDs and mesoporous film, which reducing the efficiency of CdSe QDSSCs with double ZnS barrier layers. Applying a voltage of 2 V in successive ionic layer adsorption reaction (VASILAR) to create an electric field, which assists Cd2+ and SeSO32- ions rapidly diffuse into the TiO2/ZnS mesoporous film to react forming CdSe QDs at room temperature. Optimizing the number of CdSe QDs deposition layers and combine with ZnS double-layer barrier structure, a best PCE of 4.34% for ZnS/CdSe/ZnS QDSSCs is achieved. This study gives a fast and simple approach to inhibit interfacial charge recombination to construct high performance CdSe QDSSCs.

Unraveling Heterogeneity in Transcriptome and Its Regulation Through Single-Cell Multi-Omics Technologies.

Cellular heterogeneity plays a pivotal role in tissue homeostasis and the disease development of multicellular organisms. To deconstruct the heterogeneity, a multitude of single-cell toolkits measuring various cellular contents, including genome, transcriptome, epigenome, and proteome, have been developed. More recently, multi-omics single-cell techniques enable the capture of molecular footprints with a higher resolution by simultaneously profiling various cellular contents within an individual cell. Integrative analysis of multi-omics datasets unravels the relationships between cellular modalities, builds sophisticated regulatory networks, and provides a holistic view of the cell state. In this review, we summarize the major developments in the single-cell field and review the current state-of-the-art single-cell multi-omic techniques and the bioinformatic tools for integrative analysis.

Defining Essential Enhancers for Pluripotent Stem Cells Using a Features-Oriented CRISPR-Cas9 Screen.

cis-regulatory elements (CREs) regulate the expression of genes in their genomic neighborhoods and influence cellular processes such as cell-fate maintenance and differentiation. To date, there remain major gaps in the functional characterization of CREs and the identification of their target genes in the cellular native environment. In this study, we perform a features-oriented CRISPR-utilized systematic (FOCUS) screen of OCT4-bound CREs using CRISPR-Cas9 to identify functional enhancers important for pluripotency maintenance in mESCs. From the initial 235 candidates tested, 16 CREs are identified to be essential stem cell enhancers. Using RNA-seq and genomic 4C-seq, we further uncover a complex network of candidate CREs and their downstream target genes, which supports the growth and self-renewal of mESCs. Notably, an essential enhancer, CRE111, and its target, Lrrc31, form the important switch to modulate the LIF-JAK1-STAT3 signaling pathway.

The specific immune response to tumor antigen CP1 and its correlation with improved survival in colon cancer patients.

BACKGROUND & AIMS: The present study was undertaken to determine the expression of a newly identified tumor antigen cancer-placenta 1 (CP1) in colorectal carcinoma (CRC) and explore the CP1-specific immune response in CRC patients and its correlation with patient survival. METHODS: CP1 expression was determined by reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blot analysis. Serum antibodies against CP1 were detected by enzyme-linked immunosorbent assay, and T-cell response was measured by interferon-gamma/granzyme-B release enzyme-linked immunospot assays. The HLA-A2-restricted epitopes in CP1 were predicted by bioinformatics and then experimentally validated by enzyme-linked immunospot assay. RESULTS: CP1 expression was detected in a significant number of CRC tissues, reaching 47.6% at the messenger RNA (mRNA) level and 28.6% at the protein level. Of patients with CP1 mRNA(+) tumors, more than 50% had CP1-responsive CD4(+) and CD8(+) T cells and 30% spontaneous-occurring antibodies against CP1. Further studies revealed 2 dominant HLA-A2-restricted epitopes in the CP1 antigen: p31-39 and p58-66. In a follow-up study up to 33 months after surgery, 9 of the 10 patients with CP1-specific CD8 T-cell response survived, whereas 6 of the 8 nonresponders died. Kaplan-Meier analysis indicated a significant correlation between T-cell response and patient survival. CONCLUSIONS: CP1 represents a new class of tumor-specific shared antigen. Its high expression in CRC tissues, prevalence of CP1-specific immune responses in CP1 mRNA(+) CRC patients, and positive correlation with survival suggest that the antigen may be a useful target for cancer immunotherapy.

The immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4(+) T cells, CD8(+), CD3(-)CD56(+), CD3(+)CD56(+), and gammadeltaT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3(+)CD56(+) cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4(+) cells in the tumor tended to produce more IL-10 but less IFN-gamma, whereas CD8(+) cells showed impaired capacity for the production of both IFN-gamma and perforin. Consistent with previous reports, we observed a significant increase of Foxp3(+) cells in the tumor tissue. Intriguingly, although over 90% of CD4(+)CD25(high) cells were found to be Foxp3(+), the majority of Foxp3(+) cells were identified in the CD4(+)CD25(medium) and CD4(+)CD25(-) subsets. In support of its role as a negative regulator, CD4(+)CD25(high) cells suppressed the proliferation of CD4(+)CD25(-) cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587.

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A( *)0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them-p248-256, p140-149 and p144-152-were found to bind to HLA-A(*)0201 molecules with a high affinity and effectively induced a recall response of CD8+ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587+ tumors. Notably, these peptide-specific CD8+ T cells exhibited potent cytotoxic activity over HCA587+ tumor cells. Taken together, the present study has identified three new HLA-A(*)0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.