Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Investigating the Metabolic Heterogeneity of Cancer Cells Using Functional Single-Cell Selection and nLC Combined with Multinozzle Emitter Mass Spectrometry.

Tumor metastasis and cancer recurrence are often a result of cell heterogeneity, where specific subpopulations of tumor cells may be resistant to radio- or chemotherapy. To investigate this physiological and phenotypic diversity, single-cell metabolomics provides a powerful approach at the chemical level, where distinct lipid profiles can be found in different tumor cells. Here, we established a highly sensitive platform using nanoflow liquid chromatography (nLC) combined with multinozzle emitter electrospray ionization mass spectrometry for more in-depth metabolomics profiling. Our platform identified 15 and 17 lipids from individual osteosarcoma (U2OS) and glioblastoma (GBM) cells when analyzing single-cell samples. Additionally, we used the functional single-cell selection (fSCS) pipeline to analyze the subpopulations of cells with a DNA damage response (DDR) in U2OS cells and fast migration in GBM cells. Specifically, we observed a down-regulation of polyunsaturated fatty acids (PUFAs) in U2OS cells undergoing DDR, such as fatty acids FA 20:3; O2 and FA 17:4; O3. Furthermore, ceramides (Cer 38:0; O3) and triglycerides (TG 36:0) were found to be down-regulated in fast-migrating GBM cells compared to the slow-migrating subpopulation. These findings suggest the potential roles of these metabolites and/or lipids in the cellular behavior of the subpopulations.

Quorum Sensing Is Required for the Colony Establishment of a Plant Phyllosphere Bacterium Rhodopseudomonas palustris Strain GJ-22.

The phyllosphere presents a hostile environment for many biocontrol agents; however, it is as significant as is the rhizosphere for plant health. Deploying biocontrol bacteria into the phyllosphere can efficiently suppress diseases; however, the lack of knowledge on the phyllosphere adaptive traits of biocontrol bacteria poses challenges. In this study, we demonstrated that Rhodopseudomonas palustris GJ-22 colonizes the phyllosphere by forming cell aggregates. The formation of cell aggregates required the production of exopolysaccharides (EPS), which depended on the function of the rpaI-rpaR quorum sensing (QS) mechanism, mediated by the signaling molecule p-coumaroyl-HSL (pC-HSL). The mutation of the EPS biosynthesis gene Exop1 or the signaling molecule biosynthesis gene rpaI compromised the ability of GJ-22 to tolerate reactive oxygen intermediates (ROIs), such as H2O2, in vitro and to form cell aggregates in vivo. Collectively, the results revealed that QS mediates EPS production and consequently leads to bacterial cell aggregation. IMPORTANCE Quorum sensing is used by various bacteria for coordinating the multiplication of bacterial cells in a group and for modulating the behaviors of surrounding microbial species. Host plants can benefit from this interspecies modulation, as it can disrupt the QS circuits of pathogenic bacteria. Some N-acyl homoserine lactone- (AHL-) producing bacteria that were introduced into the phyllosphere as biocontrol agents may establish AHL-based crosstalk with indigenous microbes to steer the nutritional and microecological conditions toward their own and the host plant's benefit. Here, we showed that biocontrol bacteria introduced into the phyllosphere require a functioning QS circuit to establish colonies and suppress pathogens. Furthermore, our findings provoked a broader investigation into the role of the QS circuit in beneficial microorganism-plant interactions.

A Sensitive Method for Detecting Beauveria bassiana, an Insecticidal Biocontrol Agent, Population Dynamics, and Stability in Different Substrates.

Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.

Epigenetic promoter alterations in GI tumour immune-editing and resistance to immune checkpoint inhibition.

OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.

Nivolumab versus sorafenib in advanced hepatocellular carcinoma (CheckMate 459): a randomised, multicentre, open-label, phase 3 trial.

BACKGROUND: Single-agent nivolumab showed durable responses, manageable safety, and promising survival in patients with advanced hepatocellular carcinoma in the phase 1-2 CheckMate 040 study. We aimed to investigate nivolumab monotherapy compared with sorafenib monotherapy in the first-line setting for patients with advanced hepatocellular carcinoma. METHODS: In this randomised, open-label, phase 3 trial done at medical centres across 22 countries and territories in Asia, Australasia, Europe, and North America, patients at least 18 years old with histologically confirmed advanced hepatocellular carcinoma not eligible for, or whose disease had progressed after, surgery or locoregional treatment; with no previous systemic therapy for hepatocellular carcinoma, with Child-Pugh class A and Eastern Cooperative Oncology Group performance status score of 0 or 1, and regardless of viral hepatitis status were randomly assigned (1:1) via an interactive voice response system to receive nivolumab (240 mg intravenously every 2 weeks) or sorafenib (400 mg orally twice daily) until disease progression or unacceptable toxicity. The primary endpoint was overall survival assessed in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study drug. This completed trial is registered with ClinicalTrials.gov, NCT02576509. FINDINGS: Between Jan 11, 2016, and May 24, 2017, 743 patients were randomly assigned to treatment (nivolumab, n=371; sorafenib, n=372). At the primary analysis, the median follow-up for overall survival was 15·2 months (IQR 5·7-28·0) for the nivolumab group and 13·4 months (5·7-25·9) in the sorafenib group. Median overall survival was 16·4 months (95% CI 13·9-18·4) with nivolumab and 14·7 months (11·9-17·2) with sorafenib (hazard ratio 0·85 [95% CI 0·72-1·02]; p=0·075; minimum follow-up 22·8 months); the protocol-defined significance level of p=0·0419 was not reached. The most common grade 3 or worse treatment-related adverse events were palmar-plantar erythrodysaesthesia (1 [<1%] of 367 patients in the nivolumab group vs 52 [14%] of patients in the sorafenib group), aspartate aminotransferase increase (22 [6%] vs 13 [4%]), and hypertension (0 vs 26 [7%]). Serious treatment-related adverse events were reported in 43 (12%) patients receiving nivolumab and 39 (11%) patients receiving sorafenib. Four deaths in the nivolumab group and one death in the sorafenib group were assessed as treatment related. INTERPRETATION: First-line nivolumab treatment did not significantly improve overall survival compared with sorafenib, but clinical activity and a favourable safety profile were observed in patients with advanced hepatocellular carcinoma. Thus, nivolumab might be considered a therapeutic option for patients in whom tyrosine kinase inhibitors and antiangiogenic drugs are contraindicated or have substantial risks. FUNDING: Bristol Myers Squibb in collaboration with Ono Pharmaceutical.

A phase 1b study of OXIRI in pancreatic adenocarcinoma patients and its immunomodulatory effects.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and debilitating disease with limited therapeutic options. The aim of this clinical study was to evaluate the safety, efficacy and pharmacokinetics of a novel regimen comprised of metronomic oxaliplatin (O), chronomodulated capecitabine (X) and UGT1A1 genotype-guided dosing of irinotecan (IRI) [OXIRI] as well as its immunomodulatory effects. Thirty-six patients were enrolled into either dose-escalation or expansion cohorts. In the dose escalation phase, capecitabine doses (2000, 2650, 3500 and 4500 mg/day) were administered at midnight on days 1 to 14 while oxaliplatin and irinotecan were intravenously infused at fixed doses of 50 and 75 mg/m2 respectively on days 1, 8 in a 21-day cycle. The maximum tolerated dose of capecitabine was 2650 mg/day and the most common grade 3 adverse events were neutropenia (30.6%) and diarrhea (13.9%). No grade 4 toxicity was observed. UGT1A1-genotype directed dosing resulted in similar exposure levels of irinotecan, SN-38 and SN-38G in all patients. Objective response rate was 22.2%. Median overall survival and progression-free survival were 8.1 and 5.2 months, respectively. Exploratory immunoprofiling by flow cytometry and quantitative spatial localization analysis of infiltrated immune cells performed on biopsy and plasma samples revealed significant declines in CCL22, CCL2 and TNFα levels at end of first cycle and an active host immune response. Our study showed that OXIRI was well-tolerated and exhibited good efficacy, with immunomodulatory effects. It may be considered as an alternative to FOLFIRINOX in patients intolerant to the latter.

Uncoupling immune trajectories of response and adverse events from anti-PD-1 immunotherapy in hepatocellular carcinoma.

BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.

Developing a shortened version of the dementia knowledge assessment scale (DKAS-TC) with a sample in Taiwan: an item response theory approach.

BACKGROUND: The 25-item Dementia Knowledge Assessment Scale (DKAS2) is a widely used tool for measuring knowledge of dementia. To increase the applicability of the Chinese-language version of the tool (DKAS-TC) for the general public, this study aimed to develop a shortened version using the item response theory (IRT) approach. METHODS: A total of 401 participants voluntarily completed a Chinese-language version of the DKAS2 questionnaire (DKAS-TC) at the start of dementia awareness training courses in 2020 and 2021. The four Rasch family models were used to analyze the dimensionality of the shortened scale (the DKAS-s) and to confirm its accuracy in measuring dementia knowledge. RESULTS: The results justified supported the use of a dichotomous response scale for responding to the DKAS-s and demonstrated good fit of the data to a Rasch model with the four dimensions of "Causes and Characteristics", "Communication and Engagement", "Care Needs", and "Risks and Health Promotion". Moreover, we shortened the DKAS-TC by selecting items that had both above-average discriminative ability and above-average information. The DKAS-s retained 64.13% of the information contained in the DKAS-TC, resulting in a 16-item scale which retained four items in each of the original four dimensions. The DKAS-s also correlated highly (≥0.95) with the DKAS-TC and exhibited a sizeable range of difficulty of dementia knowledge. CONCLUSIONS: The DKAS-s is expected to be more efficient in field settings while retaining an acceptable level of psychometric properties when used as a survey instrument to measure the general public's knowledge of dementia.

CheckMate 040 cohort 5: A phase I/II study of nivolumab in patients with advanced hepatocellular carcinoma and Child-Pugh B cirrhosis.

BACKGROUND & AIMS: Patients with advanced hepatocellular carcinoma (aHCC) and Child-Pugh B liver function are often excluded from clinical trials. In previous studies, overall survival for these patients treated with sorafenib was ∼3-5 months; thus, new treatments are needed. Nivolumab, alone or in combination with ipilimumab, is conditionally approved in the United States to treat patients with aHCC who previously received sorafenib. We describe nivolumab monotherapy outcomes in patients with Child-Pugh B status. METHODS: This phase I/II, open-label, non-comparative, multicentre trial (27 centres) included patients with Child-Pugh B (B7-B8) aHCC. Patients received intravenous nivolumab 240 mg every 2 weeks until unacceptable toxicity or disease progression. Primary endpoints were objective response rate (ORR) by investigator assessment (using Response Evaluation Criteria in Solid Tumors v1.1) and duration of response. Safety was assessed using National Cancer Institute Common Terminology Criteria for Adverse Events v4.0. RESULTS: Twenty-five sorafenib-naive and 24 sorafenib-treated patients began treatment between November 2016 and October 2017 (median follow-up, 16.3 months). Investigator-assessed ORR was 12% (95% CI 5-25%) with 6 patients responding; disease control rate was 55% (95% CI 40-69%). Median time to response was 2.7 months (interquartile range, 1.4-4.2), and median duration of response was 9.9 months (95% CI 9.7-9.9). Treatment-related adverse events (TRAEs) were reported in 25 patients (51%) and led to discontinuation in 2 patients (4%). The most frequent grade 3/4 TRAEs were hypertransaminasemia (n = 2), amylase increase (n = 2), and aspartate aminotransferase increase (n = 2). The safety of nivolumab was comparable to that in patients with Child-Pugh A aHCC. CONCLUSIONS: Nivolumab showed clinical activity and favourable safety with manageable toxicities, suggesting it could be suitable for patients with Child-Pugh B aHCC. LAY SUMMARY: In patients with advanced hepatocellular carcinoma, almost all systemic therapies require very good liver function, i.e. Child-Pugh A status. The evidence from this study suggests that nivolumab shows clinical activity and an acceptable safety profile in patients with hepatocellular carcinoma with Child-Pugh B status who have mild to moderate impairment of liver function or liver decompensation that might rule out other therapies. Further studies are warranted to assess the safety and efficacy of nivolumab in this patient population. CLINICAL TRIAL NUMBER: NCT01658878.

Rhodopseudomonas palustris Quorum Sensing Molecule pC-HSL Induces Systemic Resistance to TMV Infection via Upregulation of NbSIPK/NbWIPK Expressions in Nicotiana benthamiana.

G-negative bacteria produce myriad N-acyl-homoserine lactones (AHLs) that can function as quorum sensing (QS) signaling molecules. AHLs are also known to regulate various plant biological activities. p-Coumaroyl-homoserine lactone (pC-HSL) is the only QS molecule produced by a photosynthetic bacterium, Rhodopseudomonas palustris. The role of pC-HSL in the interaction between R. palustris and plant has not been investigated. In this study, we investigated the effect of pC-HSL on plant immunity and found that this QS molecule can induce a systemic resistance to Tobacco mosaic virus (TMV) infection in Nicotiana benthamiana. The results show that pC-HSL treatment can prolong the activation of two mitogen-associated protein kinase genes (i.e., NbSIPK and NbWIPK) and increase the expression of transcription factor WRKY8 as well as immune response marker genes NbPR1 and NbPR10, leading to an increased accumulation of reactive oxygen species (ROS) in the TMV-infected plants. Our results also show that pC-HSL treatment can increase activities of two ROS-scavenging enzymes, peroxidase and superoxide dismutase. Knockdown of NbSIPK or NbWIPK expression in N. benthamiana plants through virus-induced gene silencing nullified or attenuated pC-HSL-induced systemic resistance, indicating that the functioning of pC-HSL relies on the activity of those two kinases. Meanwhile, pC-HSL-pretreated plants also showed a strong induction of kinase activities of NbSIPK and NbWIPK after TMV inoculation. Taken together, our results demonstrate that pC-HSL treatment increases plant resistance to TMV infection, which is helpful to uncover the outcome of interaction between R. palustris and its host plants.

Challenges of combination therapy with immune checkpoint inhibitors for hepatocellular carcinoma.

Immune checkpoint inhibitor (ICI) therapy targeting anti-programmed cell death-1 (anti-PD-1) or its ligand (anti-PD-L1) is the backbone of numerous combination regimens aimed at improving the objective response and survival of patients with hepatocellular carcinoma (HCC). Clinical trials of immuno-oncology regimens in other cancer types have shed light on issues of study design, including how to choose candidate regimens based on early-phase trial results, statistical considerations in trials with multiple primary endpoints, and the importance of predictive biomarkers. In this review, the updated data from early-phase trials of combination immunotherapy for HCC are summarised. Since the most extensively tested combination regimens for advanced HCC comprise anti-PD-1/anti-PD-L1 agents plus antiangiogenic agents, the relative benefit and antitumor mechanism of antiangiogenic multikinase inhibitors versus specific VEGF/VEGFR inhibitors are discussed. Other critical issues in the development of combination immunotherapy, including optimal management of immune-related adverse events and the value of ICI therapy in combination with locoregional treatment for HCC, are also explored.

Development and validation of the prenatal activity restriction stress questionnaire: a Rasch rating scale analysis.

BACKGROUND: Antepartum activity restriction (AAR) is a common recommendation given to women at risk for preterm delivery. However, such treatment has been shown to cause heavy emotional burdens on the women receiving it since it requires them to face many challenges derived from the intervention. Nevertheless, current existing scales lack effective items that can reflect the distress of these women. The aim of this study was to develop a reliable instrument to assess the distress of women experiencing AAR. METHOD: The Prenatal Activity Restriction Stress Questionnaire (PARSQ) was developed according to comprehensive literature review, women's interviews, and existing pregnancy-special stress scales from August 2016 to July 2017 in southern Taiwan. Six experts evaluated its content validity; the Rasch rating scale model (RSM) was used to examine its item-fit, dimensionality, and reliability with 200 women with AAR experience. Furthermore, the concurrent validity was assessed through computing the correlation of AAR women's scores on the PARSQ and Perceived Stress Scale (PSS), and discriminant validity of the PARSQ was assessed to compare the scores' differences between the AAR women and the healthy pregnant women. RESULTS: The PARSQ was constructed with 23 items in the 4-dimensional scale: Role function changes (8 items), Fetal safety and health (5 items), Physical and psychological care issues (5 items), and Socioeconomic and medical issues (5 items). It was confirmed to have satisfactory content vitality (CVI = 0.78 to 1.0), reasonable item-fit (0.77 to 1.51), and good reliability in RSM model, as well as adequate concurrent validity (p = 0.005) and discriminant validity (p < 0.001). CONCLUSIONS: Understanding the distress of women undergoing AAR is necessary for developing appropriate prenatal care to assist women in coping with their situation to alleviate their emotional burdens. The developed PARSQ with satisfied psychometric properties can be an informative instrument for clinicians/researchers to assess the specific stress of pregnant women with AAR.

Different MicroRNA Families Involved in Regulating High Temperature Stress Response during Cotton (Gossypium hirsutum L.) Anther Development.

MicroRNAs (miRNAs) are small molecule RNAs widely involved in responses to plant abiotic stresses. We performed small RNA sequencing of cotton anthers at four developmental stages under normal and high temperature (NT and HT, respectively) conditions to investigate the stress response characteristics of miRNA to HT. A total of 77 miRNAs, including 33 known miRNAs and 44 novel miRNAs, were identified, and 41 and 28 miRNAs were differentially expressed under NT and HT stress conditions, respectively. The sporogenous cell proliferation (SCP), meiotic phase (MP), microspore release period (MRP), and pollen maturity (PM) stages had 10 (including 12 miRNAs), four (including six miRNAs), four (including five miRNAs), and seven (including 11 miRNAs) HT stress-responsive miRNA families, respectively, which were identified after removing the changes in genotype-specific miRNAs under NT condition. Seven miRNA families (miR2949, miR167, and miR160 at the SCP stage; miR156 and miR172 at the MP stage; miR156 at the MRP stage; and miR393 and miR3476 at the PM stage), which had expression abundance of more than 10% of the total expression abundance, served as the main regulators responding to HT stress with positive or negative regulation patterns. These miRNAs orchestrated the expression of the corresponding target genes and led to different responses in the HT-tolerant and the HT-sensitive lines. The results revealed that the HT stress response of miRNAs in cotton anthers were stage-specific and differed with the development of anthers. Our study may enhance the understanding of the response of miRNAs to HT stress in cotton anthers and may clarify the mechanism of plant tolerance to HT stress.

Nivolumab in advanced hepatocellular carcinoma: Sorafenib-experienced Asian cohort analysis.

BACKGROUND & AIMS: Nivolumab, an immune checkpoint inhibitor, is approved in several countries to treat sorafenib-experienced patients with HCC, based on results from the CheckMate 040 study (NCT01658878). Marked differences exist in HCC clinical presentation, aetiology, treatment patterns and outcomes across regions. This analysis assessed the safety and efficacy of nivolumab in the Asian cohort of CheckMate 040. METHODS: CheckMate 040 is an international, multicentre, open-label, phase I/II study of nivolumab in adults with advanced HCC, regardless of aetiology, not amenable to curative resection or local treatment and with/without previous sorafenib treatment. This analysis included all sorafenib-experienced patients in the intent-to-treat (ITT) overall population and Asian cohort. The analysis cut-off date was March 2018. RESULTS: There were 182 and 85 patients in the ITT population and Asian cohort, respectively. In both populations, most patients were older than 60 years, had BCLC (Barcelona Clinic Liver Cancer) Stage C disease, and had received previous systemic therapy. A higher percentage of Asian patients had HBV infections, extrahepatic metastases and prior therapies. Median follow-up was 31.6 and 31.3 months for the ITT and Asian patients, respectively. Objective response rates were 14% and 15% in the ITT population and Asian cohort, respectively. In the Asian cohort, patients with HBV, HCV or those who were uninfected had objective response rates of 13%, 14% and 21%, respectively. The median duration of response was longer in the ITT (19.4 months) vs. Asian patients (9.7 months). Median overall survival was similar between the ITT (15.1 months) and Asian patients (14.9 months), and unaffected by aetiology in Asian patients. The nivolumab safety profile was similar and manageable across both populations. CONCLUSION: Nivolumab safety and efficacy are comparable between sorafenib-experienced ITT and Asian patients. LAY SUMMARY: The CheckMate 040 study evaluated the safety and efficacy of nivolumab in patients with advanced hepatocellular carcinoma who were refractory to previous sorafenib treatment or chemotherapy. This subanalysis of the data showed that treatment responses and safety in patients in Asia were similar to those of the overall treatment population, providing support for nivolumab as a treatment option for these patients. Clinical trial number: NCT01658878.

Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial.

BACKGROUND: For patients with advanced hepatocellular carcinoma, sorafenib is the only approved drug worldwide, and outcomes remain poor. We aimed to assess the safety and efficacy of nivolumab, a programmed cell death protein-1 (PD-1) immune checkpoint inhibitor, in patients with advanced hepatocellular carcinoma with or without chronic viral hepatitis. METHODS: We did a phase 1/2, open-label, non-comparative, dose escalation and expansion trial (CheckMate 040) of nivolumab in adults (≥18 years) with histologically confirmed advanced hepatocellular carcinoma with or without hepatitis C or B (HCV or HBV) infection. Previous sorafenib treatment was allowed. A dose-escalation phase was conducted at seven hospitals or academic centres in four countries or territories (USA, Spain, Hong Kong, and Singapore) and a dose-expansion phase was conducted at an additional 39 sites in 11 countries (Canada, UK, Germany, Italy, Japan, South Korea, Taiwan). At screening, eligible patients had Child-Pugh scores of 7 or less (Child-Pugh A or B7) for the dose-escalation phase and 6 or less (Child-Pugh A) for the dose-expansion phase, and an Eastern Cooperative Oncology Group performance status of 1 or less. Patients with HBV infection had to be receiving effective antiviral therapy (viral load <100 IU/mL); antiviral therapy was not required for patients with HCV infection. We excluded patients previously treated with an agent targeting T-cell costimulation or checkpoint pathways. Patients received intravenous nivolumab 0·1-10 mg/kg every 2 weeks in the dose-escalation phase (3+3 design). Nivolumab 3 mg/kg was given every 2 weeks in the dose-expansion phase to patients in four cohorts: sorafenib untreated or intolerant without viral hepatitis, sorafenib progressor without viral hepatitis, HCV infected, and HBV infected. Primary endpoints were safety and tolerability for the escalation phase and objective response rate (Response Evaluation Criteria In Solid Tumors version 1.1) for the expansion phase. This study is registered with ClinicalTrials.gov, number NCT01658878. FINDINGS: Between Nov 26, 2012, and Aug 8, 2016, 262 eligible patients were treated (48 patients in the dose-escalation phase and 214 in the dose-expansion phase). 202 (77%) of 262 patients have completed treatment and follow-up is ongoing. During dose escalation, nivolumab showed a manageable safety profile, including acceptable tolerability. In this phase, 46 (96%) of 48 patients discontinued treatment, 42 (88%) due to disease progression. Incidence of treatment-related adverse events did not seem to be associated with dose and no maximum tolerated dose was reached. 12 (25%) of 48 patients had grade 3/4 treatment-related adverse events. Three (6%) patients had treatment-related serious adverse events (pemphigoid, adrenal insufficiency, liver disorder). 30 (63%) of 48 patients in the dose-escalation phase died (not determined to be related to nivolumab therapy). Nivolumab 3 mg/kg was chosen for dose expansion. The objective response rate was 20% (95% CI 15-26) in patients treated with nivolumab 3 mg/kg in the dose-expansion phase and 15% (95% CI 6-28) in the dose-escalation phase. INTERPRETATION: Nivolumab had a manageable safety profile and no new signals were observed in patients with advanced hepatocellular carcinoma. Durable objective responses show the potential of nivolumab for treatment of advanced hepatocellular carcinoma. FUNDING: Bristol-Myers Squibb.

Insights into the cotton anther development through association analysis of transcriptomic and small RNA sequencing.

BACKGROUND: Plant anther development is a systematic and complex process precisely controlled by genes. Regulation genes and their regulatory mechanisms for this process remain elusive. In contrast to numerous researches on anther development with respect to mRNAs or miRNAs in many crops, the association analysis combining both omics has not been reported on cotton anther. RESULTS: In this study, the molecular mechanism of cotton anther development was investigated with the employment of association analysis of transcriptome and small RNA sequencing during the predefined four stages of cotton anther development, sporogenuous cell proliferation (SCP), meiotic phase (MP), microspore release period (MRP) and pollen maturity (PM). Analysis revealed that the differentially expressed genes are increasingly recruited along with the developmental progress. Expression of functional genes differed significantly among developmental stages. The genes related with cell cycle, progesterone-mediated oocyte maturation, and meiosis are predominantly expressed at the early stage of anther development (SCP and MP), and the expression of genes involved in energy metabolism, flavonoid biosynthesis, axon guidance and phospholipase D signaling pathways is mainly enriched at the late stage of anther development (MRP and PM). Analysis of expression patterns revealed that there was the largest number of differentially expressed genes in the MP and the expression profiles of differentially expressed genes were significantly increased, which implied the importance of MP in the entire anther development cycle. In addition, prediction and analysis of miRNA targeted genes suggested that miRNAs play important roles in anther development. The miRNAs ghr-miR393, Dt_chr12_6065 and At_chr9_3080 participated in cell cycle, carbohydrate metabolism and auxin anabolism through the target genes, respectively, to achieve the regulation of anther development. CONCLUSIONS: Through the association analysis of mRNA and miRNA, our work gives a better understanding of the preferentially expressed genes and regulation in different developmental stages of cotton anther and the importance of meiotic phase, and also the involvement of miRNAs in precise regulation for this process, which would be valuable for clarifying the mechanism of plant anther development in response to internal and external environments.

HNA-3a and HNA-3b antigens among 9 ethnic populations and the Han population in Southwest China.

BACKGROUND: Human neutrophil antigen 3 (HNA-3) is encoded by the SLC44A2 gene. Antibodies against HNAs can cause severe, often fatal, transfusion reactions, known as transfusion-related acute lung injury, and neonatal neutropenia. We explored the 2 common HNA-3 variants in 9 ethnic populations residing in Sichuan and Yunnan provinces of China as compared to the Han population. METHODS: We genotyped for SLC44A2 (rs2288904) by polymerase chain reaction sequence-based typing among blood donors, for a total of 2206 individuals in Yunnan and 376 in Sichuan. RESULTS: The SLC44A2*02 allele (HNA-3b antigen) frequency varied between 0.24 and 0.33 for all 9 ethnic populations in Yunnan, including Zhuang, Derung, Hani, Lisu, Bai, Miao, Dai, Naxi, and Yi. Specifically, the Yi ethnicity did not present an unusually great SLC44A2*02 frequency at any of the 4 locations examined in Yunnan. Except of the Yi ethnicity in Sichuan (0.40), the Han ethnicity, as the majority population group, had the greatest SLC44A2*02 frequency with 0.39 in Yunnan and 0.35 in Sichuan. CONCLUSION: The ethnic populations in Southwest China are not at an increased risk for anti-HNA3a compared to the Han population, with the possible exception of Yi in Sichuan. Our data, however, corroborated the known high prevalence of SLC44A2*02 in Han populations. Hence, the Han populations in Yunnan, Sichuan and elsewhere in China are at a comparatively great risk for developing HNA-3a antibodies.

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.