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Abstract Background Previous investigations of transcriptomic signatures of cancer patient survival and post-therapy relapse have focused on tumor tissue. In contrast, here we show that in colorectal cancer (CRC) transcriptomes derived from normal tissues adjacent to tumors (NATs) are better predictors of relapse. Results Using the transcriptomes of paired tumor and NAT specimens from 80 Korean CRC patients retrospectively determined to be in recurrence or nonrecurrence states, we found that, when comparing recurrent with nonrecurrent samples, NATs exhibit a greater number of differentially expressed genes (DEGs) than tumors. Training two prognostic elastic net-based machine learning models—NAT-based and tumor-based in our Samsung Medical Center (SMC) cohort, we found that NAT-based model performed better in predicting the survival when the model was applied to the tumor-derived transcriptomes of an independent cohort of 450 COAD patients in TCGA. Furthermore, compositions of tumor-infiltrating immune cells in NATs were found to have better prognostic capability than in tumors. We also confirmed through Cox regression analysis that in both SMC-CRC as well as in TCGA-COAD cohorts, a greater proportion of genes exhibited significant hazard ratio when NAT-derived transcriptome was used compared to when tumor-derived transcriptome was used. Conclusions Taken together, our results strongly suggest that NAT-derived transcriptomes and immune cell composition of CRC are better predictors of patient survival and tumor recurrence than the primary tumor.

Summary: Predicting colorectal cancer recurrence after tumor resection is crucial because it promotes the administration of proper subsequent treatment or management to improve the clinical outcomes of patients. Several clinical or molecular factors, including tumor stage, metastasis, and microsatellite instability status, have been used to assess the risk of recurrence, although their predictive ability is limited. Here, we predicted colorectal cancer recurrence based on cellular deconvolution of bulk tumors into two distinct immune cell states: cancer-associated (tumor-infiltrating immune cell-like) and noncancer-associated (peripheral blood mononuclear cell-like). Prediction model performed significantly better when immune cells were deconvoluted into two states rather than a single state, suggesting that the difference in cancer recurrence was better explained by distinct states of immune cells. It indicates the importance of distinguishing immune cell states using cellular deconvolution to improve the prediction of colorectal cancer recurrence.

Abstract Background The 2016 World Health Organization (WHO) classification introduced nodal lymphomas of T follicular helper (Tfh) cell origin, such as angioimmunoblastic T-cell lymphoma (AITL), follicular peripheral T-cell lymphoma (F-PTCL), and nodal peripheral T-cell lymphoma with T follicular helper phenotype (nodal PTCL with TFH phenotype). However, the accurate incidence rate and clinical characteristics of F-PTCL and nodal PTCL with TFH are unstudied. Methods Between February 2012 to June 2020, a total of 207 cases diagnosed with nodal lymphomas of T follicular helper (Tfh) cell origin and PTCL-NOS were reviewed for clinical and histopathologic data. PTCL-NOS was defined to not correlate to any of the specific entities of mature T cell lymphoma in the WHO 2016 classification. We attempted to classify PTCL-GATA3 and PTCL-TBX21 by IHC staining. Target gene analysis was performed on a few patients with sufficient blood and tissue samples additionally. Results Among 207 patients, 111 patients (53.6%) had AITL, 67 patients (32.4%) had PTCL-NOS, 19 patients (9.2%) had F-PTCL, and 10 patients (4.8%) had nodal PTCL with TFH phenotype. We re-defined and analyzed F-PTCL and nodal PTCL with TFH phenotype into other TFH lymphomas. AITL (N = 101/111, 91.0%) was found to have a higher frequency of stage III/IV cancers compared to other TFH lymphomas (N = 22/29, 75.0%) and PTCL-NOS (N = 53/67, 79.1%; p-value = 0.03). The OS of AITL and other TFH lymphomas was similarly superior to PTCL-NOS (p-value = 0.02). AITL and other TFH lymphomas showed the TBX21 subtype more commonly than the GATA3 subtype. Mutations related to the RAS family (RHOA) and those related to epigenetic regulators (IDH2, DNMT3A, and TET2) were shown mainly in AITL and other TFH lymphomas. Conclusions Other TFH lymphomas appear to be a rare disease entity around one-quarter in nodal lymphomas of T follicular helper (Tfh) cell origin. Their less aggressive clinical feature than we did not expect is utterly different from PTCL-NOS and AITL. On the other hand, other TFH lymphomas share some characteristics, such as the cell of origin, a more common TBX21 subtype, and genetic variation such as RAS family mutation and epigenetic regulators, with AITL.

Pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in tumor growth, metastasis, and immune evasion in the pancreatic tumor microenvironment, and recent studies suggest an association between PAUF expression and poor prognosis in ovarian cancer patients. The current study aimed 1) to characterize the potential tumor-promoting role of PAUF in ovarian cancer, using in vitro and in vivo models, including a PAUF-knockout OVCAR-5 cell line, and 2) to explore the potential therapeutic effects of an anti-PAUF antibody for ovarian cancer. Recombinant PAUF significantly increased tumor metastatic capacity (migration, invasion, and adhesion) in all the ovarian cancer cell lines tested, except for the OVCAR-5 cell line which expresses PAUF at a much higher level than the other cells. PAUF-knockout in the OVCAR-5 cell line led to apparently delayed tumor growth in vitro and in vivo. Furthermore, the administration of an anti-PAUF antibody exhibited notable sensitizing and synchronizing effects on docetaxel in mice bearing the OVCAR-5 xenograft tumors. Taken together, this study shows that the expression level of PAUF is an independent factor determining malignant behaviors of ovarian cancer and, for the first time, it suggests that PAUF may be a promising therapeutic target for high PAUF-expressing ovarian cancer.

Abstract We evaluated the predictive role of circulating tumor DNA (ctDNA) detection by targeted deep sequencing in patients with metastatic renal cell carcinoma (mRCC) treated with immune checkpoint blockades (ICB). To determine the feasibility of ctDNA detection in our panel encompassing 40 genes, we collected 10 ml of blood from 20 patients at the time of radical nephrectomy. We analyzed somatic mutations in primary tumors and ctDNA samples from these patients. We finally collected 10 ml of blood before and after 1 month of treatment, respectively, from four patients with mRCC who received first-line ICB treatment. Variants were detected in primary tumors of 15 patients (75%) and ctDNA was detected in the plasma of 9 patients (45%). We examined the predictive role of ctDNA in four patients who received first-line ICB therapy. In two patients showing partial response, ctDNA levels decreased after 1 month of ICB treatment. However, in one patient who showed disease progression, ctDNA levels increased after 1 month of ICB treatment. Taken together, ctDNA detection in plasma by targeted deep sequencing was feasible in patients with RCC. Moreover, the levels of ctDNA could be an early predictor of treatment response in patients with mRCC who receive ICB therapy.

Hypertension is a major, yet manageable, risk factor for stroke, and the benefits of well-controlled blood pressure are well established. However, the strategy for managing blood pressure can differ based on the pathomechanism (subtype), stage, and treatment of stroke patients. In the present review, we focused on the management of blood pressure during the acute stage of intracerebral hemorrhage, subarachnoid hemorrhage, and cerebral infarction. In patients with cerebral infarction, the target blood pressure was discussed both before and after thrombolysis or other endovascular treatment, which may be an important issue. When and how to start antihypertensive medications during the acute ischemic stroke period were also discussed. In regards to the secondary prevention of ischemic stroke, the target blood pressure may differ based on the mechanism of ischemic stroke. We have reviewed previous studies and guidelines to summarize blood pressure management in various situations involving stroke patients.

Although the combination of radiotherapy and immunotherapy has proven to be effective in lung cancer treatment, it may not be sufficient to fully activate the antitumor immune response. Here, we investigated whether entinostat, a histone deacetylase inhibitor, could improve the efficacy of radiotherapy and anti-PD-1 in a murine syngeneic LL/2 tumor model. A total of 12 Gy of X-rays administered in two fractions significantly delayed tumor growth in mice, which was further enhanced by oral entinostat administration. Flow cytometry-aided immune cell profiling revealed that entinostat increased radiation-induced infiltration of myeloid-derived suppressor cells and CD8+ T cells with decreased regulatory T-cells (Tregs). Transcriptomics-based immune phenotype prediction showed that entinostat potentiated radiation-activated pathways, such as JAK/STAT3/interferon-gamma (IFN-γ) and PD-1/PD-L1 signaling. Entinostat augmented the antitumor efficacy of radiation and anti-PD-1, which may be related to an increase in IFN-γ-producing CD8+ T-cells with a decrease in Treg cells. Comparative transcriptomic profiling predicted that entinostat increased the number of dendritic cells, B cells, and T cells in tumors treated with radiation and anti-PD-1 by inducing MHC-II genes. In conclusion, our findings provided insights into how entinostat improves the efficacy of ionizing radiation plus anti-PD-1 therapy and offered clues for developing new strategies for clinical trials.

Abstract Background Seed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size. Results Proteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical β-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds. Conclusions Collectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

Abstract Background Targeted deep sequencing is increasingly used to detect low-allelic fraction variants; it is therefore essential that errors that constitute baseline noise and impose a practical limit on detection are characterized. In the present study, we systematically evaluate the extent to which errors are incurred during specific steps of the capture-based targeted sequencing process. Results We removed most sequencing artifacts by filtering out low-quality bases and then analyze the remaining background noise. By recognizing that plasma DNA is naturally fragmented to be of a size comparable to that of mono-nucleosomal DNA, we were able to identify and characterize errors that are specifically associated with acoustic shearing. Two-thirds of C:G > A:T errors and one quarter of C:G > G:C errors were attributed to the oxidation of guanine during acoustic shearing, and this was further validated by comparative experiments conducted under different shearing conditions. The acoustic shearing step also causes A > G and A > T substitutions localized to the end bases of sheared DNA fragments, indicating a probable association of these errors with DNA breakage. Finally, the hybrid selection step contributes to one-third of the remaining C:G > A:T and one-fifth of the C > T errors. Conclusions The results of this study provide a comprehensive summary of various errors incurred during targeted deep sequencing, and their underlying causes. This information will be invaluable to drive technical improvements in this sequencing method, and may increase the future usage of targeted deep sequencing methods for low-allelic fraction variant detection.

Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a PRR7::PRR7-luciferase (PRR7-LUC) translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of ELF3 and ELF4, core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of ELF3 that help to understand the relationship between molecular potency and phenotype.

Abstract Adenoviruses are commonly utilized as viral vectors for gene therapy, genetic vaccines, and recombinant protein expression. To generate replication-defective adenoviruses, E1-complementing cell lines such as HEK293A are utilized; however, limitations remain. Repeated passage of E1-deleted virus in HEK293A cells increases the occurrence of replication-competent adenoviruses (RCAs). In the present study, we developed a novel cell line originating from human primary cells. L132 cells were transduced two times with E1-encoded retrovirus and three times with E1A-encoded retrovirus. Finally, we selected the most productive L132 cell line for generation of RCA-free adenovirus, GT541. GT541 can serve as an alternative cell line to HEK293A and other adenovirus-producing cells.

Abstract Photothermal therapy is an alternative cancer therapy that uses a photothermal agent with light irradiation to induce fatal hyperthermia in cancer cells. In a previous study, we found that ex vivo photothermal (PT) treatment induced expression of heat shock proteins (HSPs), such as HSP70, HSP27, and HSP90, in cancer cells; moreover, immunization with lysates from PT-treated tumor cells resulted in significant tumor growth inhibition in tumor-bearing mice. In this study, we hypothesized that sublethal PT treatment of antigen-presenting cells regulates their immunogenicity. We observed the upregulation of expression of intracellular HSP70 and surface activation markers, such as CD40, CD80, CD86, and MHC class II, in sublethal PT-treated cells. The protumoral activity of myeloid-derived suppressor cells (MDSCs) was reduced by sublethal hyperthermia. Furthermore, poorly immunogenic MDSCs were converted into immunogenic antigen-presenting cells by PT treatment. The differences in immunogenicity between MDSCs untreated or treated with the PT technique were evaluated using the Student’s t-test or Mann–Whitney rank sum test. Collectively, direct hyperthermic treatment resulted in phenotypic changes and the functional regulation of immune cells.

Panax ginseng C.A. Meyer is a traditional medicinal herb that produces bioactive compounds such as ginsenosides. Here, we investigated the diversity of ginsenosides and related genes among five genetically fixed inbred ginseng cultivars (Chunpoong [CP], Cheongsun [CS], Gopoong [GO], Sunhyang [SH], and Sunun [SU]). To focus on the genetic diversity related to ginsenoside biosynthesis, we utilized in vitro cultured adventitious roots from the five cultivars grown under controlled environmental conditions. PCA loading plots based on secondary metabolite composition classified the five cultivars into three groups. We selected three cultivars (CS, SH, and SU) to represent the three groups and conducted further transcriptome and gas chromatography-mass spectrometry analyses to identify genes and intermediates corresponding to the variation in ginsenosides among cultivars. We quantified ginsenoside contents from the three cultivars. SH had more than 12 times the total ginsenoside content of CS, with especially large differences in the levels of panaxadiol-type ginsenosides. The expression levels of genes encoding squalene epoxidase (SQE) and dammarenediol synthase (DDS) were also significantly lower in CS than SH and SU, which is consistent with the low levels of ginsenoside produced in this cultivar. Methyl jasmonate (MeJA) treatment increased the levels of panaxadiol-type ginsenosides up to 4-, 13-, and 31-fold in SH, SU, and CS, respectively. MeJA treatment also greatly increased the quantity of major intermediates and the expression of the underlying genes in the ginsenoside biosynthesis pathway; these intermediates included squalene, 2,3-oxidosqualene, and dammarenediol II, especially in CS, which had the lowest ginsenoside content under normal culture conditions. We conclude that SQE and DDS are the most important genetic factors for ginsenoside biosynthesis with diversity among ginseng cultivars.

Abstract Background Our study aims to evaluate the genetic and phenotypic spectrum of Frontotemporal dementia (FTD) gene variant carriers in Chinese populations, investigate mutation frequencies, and assess the functional properties of TBK1 and OPTN variants. Methods Clinically diagnosed FTD patients underwent genetic analysis through exome sequencing, repeat-primed polymerase chain reaction, and Sanger sequencing. TBK1 and OPTN variants were biologically characterized in vitro using immunofluorescence, immunoprecipitation, and immunoblotting analysis. The frequencies of genes implicated in FTD in China were analyzed through a literature review and meta-analysis. Results Of the 261 Chinese FTD patients, 61 (23.4%) carried potential causative variants in FTD-related genes, including MAPT (n = 17), TBK1 (n = 7), OPTN (n = 6), GRN (n = 6), ANXA11 (n = 4), CHMP2B (n = 3), C9orf72 GGGGCC repeats (n = 2), CYLD (n = 2), PRNP (n = 2), SQSTM1 (n = 2), TARDBP (n = 2), VCP (n = 1), CCNF (n = 1), CHCHD10 (n = 1), SIGMAR1 (n = 1), CHCHD2 (n = 1), FUS (n = 1), TMEM106B (n = 1), and UBQLN2 (n = 1). 29 variants can be considered novel, including the MAPT p.D54N, p.E342K, p.R221P, p.T263I, TBK1 p.E696G, p.I37T, p.E232Q, p.S398F, p.T78A, p.Q150P, p.W259fs, OPTN p.R144G, p.F475V, GRN p.V473fs, p.C307fs, p.R101fs, CHMP2B p.K6N, p.R186Q, ANXA11 p.Q155*, CYLD p.T157I, SQSTM1 p.S403A, UBQLN2 p.P509H, CCNF p.S160N, CHCHD10 p.A8T, SIGMAR1 p.S117L, CHCHD2 p.P53fs, FUS p.S235G & p.S236G, and TMEM106B p.L144V variants. Patients with TBK1 and OPTN variants presented with heterogeneous clinical phenotypes. Functional analysis demonstrated that TBK1 I37T and E232Q mutants showed decreased autophosphorylation, and the OPTN phosphorylation was reduced by the TBK1 I37T mutant. The OPTN-TBK1 complex formation was enhanced by the TBK1 E696G mutant, while OPTN R144G and F475V mutants exhibited reduced recruitment to autophagosomes compared to the wild-type. The overall frequency of TBK1 and OPTN in Chinese FTD patients was 2.0% and 0.3%, respectively. Conclusions Our study demonstrates the extensive genetic and phenotypic heterogeneity of Chinese FTD patients. TBK1 mutations are the second most frequent cause of clinical FTD after MAPT in the Chinese.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

Aloe vera (Asphodeloideae) is a medicinal plant in which useful secondary metabolites are plentiful. Among the representative secondary metabolites of Aloe vera are the anthraquinones including aloe emodin and chrysophanol, which are tricyclic aromatic quinones synthesized via a plant-specific type III polyketide biosynthesis pathway. However, it is not yet clear which cellular responses can induce the pathway, leading to production of tricyclic aromatic quinones. In this study, we examined the effect of endogenous elicitors on the type III polyketide biosynthesis pathway and identified the metabolic changes induced in elicitor-treated Aloe vera adventitious roots. Salicylic acid, methyl jasmonate, and ethephon were used to treat Aloe vera adventitious roots cultured on MS liquid media with 0.3 mg/L IBA for 35 days. Aloe emodin and chrysophanol were remarkably increased by the SA treatment, more than 10-11 and 5-13 fold as compared with untreated control, respectively. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry analysis identified a total of 37 SA-induced compounds, including aloe emodin and chrysophanol, and 3 of the compounds were tentatively identified as tricyclic aromatic quinones. Transcript accumulation analysis of polyketide synthase genes and gas chromatography mass spectrometry showed that these secondary metabolic changes resulted from increased expression of octaketide synthase genes and decreases in malonyl-CoA, which is the precursor for the tricyclic aromatic quinone biosynthesis pathway. In addition, anti-inflammatory activity was enhanced in extracts of SA-treated adventitious roots. Our results suggest that SA has an important role in activation of the plant specific-type III polyketide biosynthetic pathway, and therefore that the efficacy of Aloe vera as medicinal agent can be improved through SA treatment.

Protein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species) scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

Amphiphysin (AMPH) autoimmunity is associated with a variety of neurological complications, including encephalitis, peripheral neuropathy, myelopathy, and cerebellar syndrome. Its diagnosis is based on clinical neurological deficits and the presence of serum anti-AMPH antibodies. Active immunotherapy, such as intravenous immunoglobulins, steroids, and other immunosuppressive therapies, has been reported to be effective in most patients. However, the extent of recovery varies depending on the case. Herein, we report the case of a 75-year-old woman with semi-rapidly progressive systemic tremors, visual hallucinations, and irritability. Upon hospitalization, she developed a mild fever and cognitive impairment. Brain magnetic resonance imaging (MRI) showed semi-rapidly progressive diffuse cerebral atrophy (DCA) over 3 months, while no clear abnormal intensities were observed. The nerve conduction study revealed sensory and motor neuropathy in the limbs. The fixed tissue-based assay (TBA) failed to detect antineuronal antibodies; however, based on commercial immunoblots, the presence of anti-AMPH antibodies was suspected. Therefore, serum immunoprecipitation was performed, which confirmed the presence of anti-AMPH antibodies. The patient also had gastric adenocarcinoma. High- dose methylprednisolone, and intravenous immunoglobulin were administered and tumor resection was performed, resulting in resolution of the cognitive impairment and improvement in the DCA on the post-treatment MRI. After immunotherapy and tumor resection, the patient's serum was analyzed using immunoprecipitation, which showed a decrease in the level of anti-AMPH antibodies. This case is noteworthy because the DCA showed improvement after immunotherapy and tumor resection. Additionally, this case demonstrates that negative TBA with positive commercial immunoblots do not necessarily indicate false positive results. [ABSTRACT FROM AUTHOR]