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Supplementary Methods; Supplementary Figures S1-S9; Supplementary Tables S1-S7

This study aimed to explore the effective strategies in comparison with instructional designs: the face-to-face and online in-class instructions of flipped learning. For this purpose, a survey was developed and the perceptions of 22 instructors and 203 students were examined. The data analysis were conducted using content analysis. The findings were as follows: First, the survey found that several commonalities pertaining to learning aspects were derived from the students’learning activities regardless of the instructional designs. However, the dissimilarity was found in the manner in which the instructors provided their feedback. Second, 8 satisfaction determinant factors, such as space-time convenience, improvement of learning performance, teaching-learning method, facilitation of in-class activities, facilitating of communication, enhancing students’understanding, repeated learning, and self-directed learning were derived from the learners’ perception analysis. In addition, learners perceived 4 main aspects as dissatisfaction determinant factors: environmental obstacles to learning, limit of understanding, lack of communication, and an excessive amount of learning. Irrespective of instructional designs, no major difference was found in the learners’ perceptions. Third, instructors commonly perceived that flipped learning classes involved 6 implemental characteristics: motivation of learning, problem-solving, application activities, feedback, facilitating communication, and follow-up instruction. Furthermore, 6 difficulties on were also registered: implementation, communication, class operation time, instructional environment, the training program, and regulations. However, some differences in details were found based on the class designs. In conclusion, implications and suggestions for implementing flipped learning classes and following studies have been discussed.

In the TRANSCEND NHL 001 study, 53% of patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with lisocabtagene maraleucel (liso-cel) achieved a complete response (CR). To determine characteristics of patients who did and did not achieve a CR, we examined the tumor biology and microenvironment from lymph node tumor biopsies. LBCL biopsies from liso-cel–treated patients were taken pretreatment and ∼11 days posttreatment for RNA sequencing (RNA-seq) and multiplex immunofluorescence (mIF). We analyzed gene expression data from pretreatment biopsies (N = 78) to identify gene sets enriched in patients who achieved a CR to those with progressive disease. Pretreatment biopsies from month-3 CR patients displayed higher expression levels of T-cell and stroma-associated genes, and lower expression of cell-cycle genes. To interpret whether LBCL samples were “follicular lymphoma (FL)–like,” we constructed an independent gene expression signature and found that patients with a higher “FL-like” gene expression score had longer progression-free survival (PFS). Cell of origin was not associated with response or PFS, but double-hit gene expression was associated with shorter PFS. The day 11 posttreatment samples (RNA-seq, N = 73; mIF, N = 53) had higher levels of chimeric antigen receptor (CAR) T-cell densities and CAR gene expression, general immune infiltration, and immune activation in patients with CR. Further, the majority of T cells in the day 11 samples were endogenous. Gene expression signatures in liso-cel–treated patients with LBCL can inform the development of combination therapies and next-generation CAR T-cell therapies.

Here, we characterized a gene expression system induced by 3-hydroxypropionic acid (3-HP) in Pseudomonas denitrificans. The system consists of a putative LysR-type transcriptional regulator (LTTR) encoded by hpdR and a LTTR-responsive promoter that positively controls the expression of hpdH, encoding 3-HP dehydrogenase in the presence of 3-HP. In the hpdH-responsive promoter region, two operators exhibiting dyad symmetry and designated O1 and O2 and centered at the −73 and −30 positions, respectively, were located upstream of the hpdH transcription start site. When either O1, O2, or both regions were mutated, the inducibility by the HpdR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcriptional activation. The HpdR protein and its operator sites on hpdH were highly specific for each other, and did not engage in cross-talk with another similar 3-HP-inducible system that is also present in P. denitrificans. This 3-HP-inducible promoter system should be useful in developing biosensors for 3-HP, and/or dynamic gene expression systems for metabolic engineering purposes.

Most patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) have exhausted their treatment options and are deemed palliative. CD19-directed chimeric antigen receptor (CAR) T-cell therapy has recently been introduced as a new option for these patients. Lisocabtagene maraleucel (liso-cel) is an investigational CAR T-cell therapy that has shown promising activity in this setting. We used an unanchored matching-adjusted indirect comparison (MAIC) methodology to compare liso-cel, using individual patient-level data from the TRANSCEND NHL 001 (TRANSCEND; NCT02631044) trial, to salvage chemotherapy, using summary-level data from the SCHOLAR-1 study, for the treatment of patients with R/R LBCL. Standardized mean differences were used to evaluate imbalances between the TRANSCEND and SCHOLAR-1 studies. MAIC was conducted to determine the relative efficacy of liso-cel vs. salvage chemotherapy with regard to overall survival, complete response rate, and objective response rate. For all efficacy outcomes assessed, comparisons of clinical factors before MAIC showed that five of seven baseline characteristics were similar between the TRANSCEND and SCHOLAR-1 studies; however, age and R/R to last therapy status differed between studies, thus requiring matching and adjusting to ensure the validity of this analysis. The base case analyses demonstrated a significantly lower risk of mortality (hazard ratio, 0.5; 95% confidence interval [CI] 0.4–0.6; p

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated high response rates in patients with relapsed/refractory large B-cell lymphoma (LBCL); however, these therapies are associated with 2 CAR T cell–specific potentially severe adverse events (AEs): cytokine release syndrome (CRS) and neurological events (NEs). This study estimated the management costs associated with CRS/NEs among patients with relapsed/refractory LBCL using data from the pivotal TRANSCEND NHL 001 trial of lisocabtagene maraleucel, an investigational CD19-directed defined composition CAR T-cell product with a 4-1BB costimulation domain administered at equal target doses of CD8+ and CD4+ CAR+ T cells. This retrospective analysis of patients from TRANSCEND with prospectively identified CRS and/or NE episodes examined relevant trial-observed health care resource utilization (HCRU) associated with toxicity management based on the severity of the event from the health care system perspective. Cost estimates for this analysis were taken from publicly available databases and published literature. Of 268 treated patients as of April 2019, 127 (47.4%) experienced all-grade CRS and/or NEs, which were predominantly grade ≤2 (77.2%). Median total AE management costs ranged from $1930 (grade 1 NE) to $177 343 (concurrent grade ≥3 CRS and NE). Key drivers of cost were facility expenses, including intensive care unit and other inpatient hospitalization lengths of stay. HCRU and costs were significantly greater among patients with grade ≥3 AEs (22.8%). Therefore, CAR T-cell therapies with a low incidence of severe CRS/NEs will likely reduce HCRU and costs associated with managing patients receiving CAR T-cell therapy. This clinical trial was registered at www.clinicaltrials.gov as #NCT02631044.

The relationship between colitis-associated colorectal cancer (CAC) and the dysregulation of iron metabolism has been implicated. However, studies on the influence of dietary iron deficiency on the incidence of CAC are limited. This study investigated the effects of dietary iron deficiency and dietary non-heme iron on CAC development in an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. The four-week-old mice were divided into the following groups: iron control (IC; 35 ppm iron/kg) + normal (NOR), IC + AOM/DSS, iron deficient (ID

Background In the absence of randomized studies directly comparing chimeric antigen receptor T cell therapies, this study used matching-adjusted indirect comparisons (MAIC) to evaluate the comparative efficacy and safety of lisocabtagene maraleucel (liso-cel) versus axicabtagene ciloleucel (axi-cel) in patients with relapsed or refractory large B cell lymphoma (LBCL). Methods Primary data sources included individual patient data from the TRANSCEND NHL 001 study (TRANSCEND [NCT02631044]; N = 256 for efficacy set, N = 269 for safety set) for liso-cel and summary-level data from the ZUMA-1 study (NCT02348216; N = 101 for efficacy set, N = 108 for safety set) for axi-cel. Inter-study differences in design, eligibility criteria, baseline characteristics, and outcomes were assessed and aligned to the extent feasible. Clinically relevant prognostic factors were adjusted in a stepwise fashion by ranked order. Since bridging therapy was allowed in TRANSCEND but not ZUMA-1, the initial efficacy and safety analyses included bridging therapy use as a matching factor (TRANSCEND patients who received bridging therapy were removed). Subsequent sensitivity analyses excluded this matching factor. Results The initial analysis showed similar MAIC-weighted efficacy outcomes between TRANSCEND and ZUMA-1 for overall and complete response rates (odds ratio [95% confidence interval (CI)], 1.40 [0.56–3.49] and 1.21 [0.56–2.64], respectively) and for overall survival and progression-free survival (hazard ratio [95% CI], 0.81 [0.44–1.49] and 0.95 [0.58–1.57], respectively). MAIC-weighted safety outcomes favored liso-cel, with significantly lower odds of all-grade and grade ≥ 3 cytokine release syndrome (odds ratio [95% CI], 0.03 [0.01–0.07] and 0.08 [0.01–0.67], respectively) and study-specific neurological events (0.16 [0.08–0.33] and 0.05 [0.02–0.15], respectively). Efficacy and safety outcomes remained similar in sensitivity analyses, which did not include use of bridging therapy as a matching factor. Conclusions After matching and adjusting for clinically relevant prognostic factors, liso-cel demonstrated comparable efficacy and a more favorable safety profile compared with axi-cel in patients with third- or later-line relapsed or refractory LBCL. Trial registration: NCT02631044 and NCT02348216

Lactic acid has two stereoisomers of D(−)- and L(+)-forms, both of which are important monomers of biodegradable plastic, poly-lactic acid. In this study, a novel d-lactate inducible system was identified in Pseudomonas fluorescens A506, partially characterized and tested as biosensor. The d-lactate catabolic operon (lldP-dld-II) was negatively regulated through the inversely transcribed D-lldR (encoding a GntR-type regulator), where the repression is relieved by addition of d-lactate. The derepression was specific to d-lactate and marginally affected by l-lactate. The D-LldR-responsive operator, showing dyad symmetry and separated by one base, was located between +11 and + 27 from the transcription start site of the lldP-dld-II operon. By site-directed mutagenesis, a motif with a dyad symmetry (AATTGGTAtTACCAATT), present in the upstream region of lldP, was identified as essential for the binding of LldR. d-lactate biosensors were developed by connecting the upregulation by d-lactate to a green fluorescent readout. About ~6.0-fold induction by 100 mM d-lactate was observed compared to l-lactate. Keywords: Lactic acid, Enantiomer, Biosensor, Repression

CD19-directed chimeric antigen receptor (CAR) T-cell therapy has shown efficacy as a third-line or later treatment in patients with relapsed/refractory large B-cell lymphoma (LBCL). Using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and the EuroQol 5-Dimension 5-Level (EQ-5D-5L) questionnaire, we evaluated the impact of CAR T-cell treatment with lisocabtagene maraleucel (liso-cel) on health-related quality of life (HRQoL) and symptoms in patients with relapsed/refractory LBCL in the ongoing, open-label, nonrandomized TRANSCEND NHL 001 trial. Clinically meaningful improvement was observed in EORTC QLQ-C30 scores for global health status/QoL, based on a minimally important difference of 10 points at 2 to 18 months after liso-cel infusion. There were no clinically meaningful changes in physical functioning and pain, whereas clinically meaningful improvements were observed in fatigue at 2, 12, and 18 months. The proportion of patients with clinically meaningful improvement in global health status/QoL was generally higher for treatment responders than for nonresponders. A trend toward decreased mean EQ-5D-5L index scores was observed at 1 month after liso-cel infusion, followed by subsequent increases through 18 months. Mean EQ-5D-5L visual analog scale scores increased from 2 through 18 months. In summary, patients with relapsed/refractory LBCL treated with liso-cel had early, sustained, and clinically meaningful improvements in HRQoL and symptoms that correlated with antitumor activity. This study was registered at www.clinicaltrials.gov as #NCT02631044.

Acetate is regarded as a promising carbon feedstock in biological production owing to its possible derivation from C1 gases such as CO, CO2 and methane. To best use of acetate, comprehensive understanding of acetate metabolisms from genes and enzymes to pathways and regulations is needed. This review aims to provide an overview on the potential of acetate as carbon feedstock for industrial biotechnology. Biochemical, microbial and biotechnological aspects of acetate metabolism are described. Especially, the current state-of-the art in the production of value-added chemicals from acetate is summarized. Challenges and future perspectives are also provided.

Summary Background Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, chimeric antigen receptor (CAR) T-cell product. We aimed to assess the activity and safety of liso-cel in patients with relapsed or refractory large B-cell lymphomas. Methods We did a seamless design study at 14 cancer centres in the USA. We enrolled adult patients (aged ≥18 years) with relapsed or refractory large B-cell lymphomas. Eligible histological subgroups included diffuse large B-cell lymphoma, high-grade B-cell lymphoma with rearrangements of MYC and either BCL2, BCL6, or both (double-hit or triple-hit lymphoma), diffuse large B-cell lymphoma transformed from any indolent lymphoma, primary mediastinal B-cell lymphoma, and follicular lymphoma grade 3B. Patients were assigned to one of three target dose levels of liso-cel as they were sequentially tested in the trial (50 × 106 CAR+ T cells [one or two doses], 100 × 106 CAR+ T cells, and 150 × 106 CAR+ T cells), which were administered as a sequential infusion of two components (CD8+ and CD4+ CAR+ T cells) at equal target doses. Primary endpoints were adverse events, dose-limiting toxicities, and the objective response rate (assessed per Lugano criteria); endpoints were assessed by an independent review committee in the efficacy-evaluable set (comprising all patients who had confirmed PET-positive disease and received at least one dose of liso-cel). This trial is registered with ClinicalTrials.gov, NCT02631044. Findings Between Jan 11, 2016, and July 5, 2019, 344 patients underwent leukapheresis for manufacture of CAR+ T cells (liso-cel), of whom 269 patients received at least one dose of liso-cel. Patients had received a median of three (range 1–8) previous lines of systemic treatment, with 260 (97%) patients having had at least two lines. 112 (42%) patients were aged 65 years or older, 181 (67%) had chemotherapy-refractory disease, and seven (3%) had secondary CNS involvement. Median follow-up for overall survival for all 344 patients who had leukapheresis was 18·8 months (95% CI 15·0–19·3). Overall safety and activity of liso-cel did not differ by dose level. The recommended target dose was 100 × 106 CAR+ T cells (50 × 106 CD8+ and 50 × 106 CD4+ CAR+ T cells). Of 256 patients included in the efficacy-evaluable set, an objective response was achieved by 186 (73%, 95% CI 66·8–78·0) patients and a complete response by 136 (53%, 46·8–59·4). The most common grade 3 or worse adverse events were neutropenia in 161 (60%) patients, anaemia in 101 (37%), and thrombocytopenia in 72 (27%). Cytokine release syndrome and neurological events occurred in 113 (42%) and 80 (30%) patients, respectively; grade 3 or worse cytokine release syndrome and neurological events occurred in six (2%) and 27 (10%) patients, respectively. Nine (6%) patients had a dose-limiting toxicity, including one patient who died from diffuse alveolar damage following a dose of 50 × 106 CAR+ T cells. Interpretation Use of liso-cel resulted in a high objective response rate, with a low incidence of grade 3 or worse cytokine release syndrome and neurological events in patients with relapsed or refractory large B-cell lymphomas, including those with diverse histological subtypes and high-risk features. Liso-cel is under further evaluation at first relapse in large B-cell lymphomas and as a treatment for other relapsed or refractory B-cell malignancies. Funding Juno Therapeutics, a Bristol-Myers Squibb Company.

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Background: Liso-cel is an autologous, CD19-directed, defined composition, 4-1BB CAR T cell product administered at equal target doses of CD8 + and CD4 + CAR + T cells. In the pivotal TRANSCEND NHL 001 study (NCT02631044) in patients (pts) with R/R LBCL after ≥ 2 prior lines of therapy, liso-cel demonstrated significant clinical activity with an objective response rate of 73%, complete response (CR) rate of 53%, and median duration of response (DOR) not reached at 12 months' median DOR follow-up, and a low incidence of severe cytokine release syndrome (2%) and neurological events (10%; Abramson et al. Lancet 2020;396:839-852). While CAR T cells have shown remarkable efficacy in treating LBCL, with a well-understood mechanism of direct CAR T cell tumor killing, the contribution of the endogenous immune system in this response is currently unclear. Previously, histologic and gene expression analysis of pre- and posttreatment tumor biopsies from pts who received liso-cel in TRANSCEND NHL 001 showed a relationship between tumor-infiltrating CAR-negative (endogenous) T cells and early and durable response [Reiss et al. Blood 2019;134(suppl 1):202]. Here we examine, by gene expression and flow cytometry, post-infusion characteristics of liso-cel and peripheral endogenous T cells in pts from TRANSCEND NHL 001 with early (CR at 1 or 3 months) and long-term (CR at 9 or 12 months) response versus pts with progressive disease (PD) before or at the same time points. Methods: CAR and endogenous T cells were assayed using a novel cell-sorting and low-input RNA-seq method. CAR and endogenous T cells were isolated from peripheral blood mononuclear cell samples from 39 pts at time to maximum concentration of liso-cel (T max; Day 8-22), 1 month, and 2 months after liso-cel infusion. In addition, samples from 44 pts were assayed only at T max by RNA-seq. A subset of these samples was assessed by flow cytometry for expression of the T cell differentiation markers CD4, CD8, CD45RA, CCR7, CD27, and CD28. All RNA-seq analyses were performed in R version 4.0.2. Differential expression analysis was performed with the DESeq2 package with gene set enrichment analysis using clusterProfiler and GSVA packages with gene sets obtained from MSigDB. Comparisons of 2 groups were made with a Wilcoxon rank sum test. Association with the time-to-event outcome of progression-free survival (PFS) was assessed using Cox proportional hazards model or log rank test. Results: Characteristics consistent with less differentiated CAR T cells and endogenous T cells were significantly associated with early and long-term response. Elevated CAR T cell expression of CD27 at T max was associated with CR at 1 month and longer PFS. Expression of CCR7 by CAR T cells at 1 and 2 months was associated with CR at 9 months. Additionally, at T max, gene sets associated with T cell effector differentiation and metabolism were enriched in CAR T cells from pts with early relapse (pts with CR vs PD at 3 months). In endogenous T cells, elevated expression of CCR7 and CD28 at T max was associated with early response (CR at 1 and 3 months). Additionally, expression of CCR7 and CD28 in T cells isolated at 1 and 2 months was associated with long-term response (CR at 9 and 12 months). Furthermore, expression of the immune checkpoint receptor LAG3 by endogenous T cells was associated with PD from month 3 to month 12. Conclusions: In summary, the data from our study show the relationship between CAR T cell differentiation status and achieving response over time, and provide new evidence for the involvement of endogenous T cells in the therapeutic efficacy of CAR T cell therapy. Disclosures Thorpe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jiang: Bristol Myers Squibb: Ended employment in the past 24 months. Rytlewski: Adaptive Biotechnologies: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Kostic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kim: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Peiser: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.