The BTLA-HVEM axis restricts CAR T cell efficacy in cancer.

The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.

Streamlined measurement of chimeric antigen receptor T-cell concentration, size, viability and two-color phenotyping during manufacturing.

BACKGROUND AIMS: The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype). METHODS: In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer). RESULTS: Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression. CONCLUSIONS: These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples.

Modulation of the gut microbiota engages antigen cross-presentation to enhance antitumor effects of CAR T cell immunotherapy.

Several studies have shown the influence of commensal microbes on T cell function, specifically in the setting of checkpoint immunotherapy for cancer. In this study, we investigated how vancomycin-induced gut microbiota dysbiosis affects chimeric antigen receptor (CAR) T immunotherapy using multiple preclinical models as well as clinical correlates. In two murine tumor models, hematopoietic CD19+-A20 lymphoma and CD19+-B16 melanoma, mice receiving vancomycin in combination with CD19-directed CAR T cell (CART-19) therapy displayed increased tumor control and tumor-associated antigens (TAAs) cross-presentation compared with CART-19 alone. Fecal microbiota transplant from human healthy donors to pre-conditioned mice recapitulated the results obtained in naive gut microbiota mice. Last, B cell acute lymphoblastic leukemia patients treated with CART-19 and exposed to oral vancomycin showed higher CART-19 peak expansion compared with unexposed patients. These results substantiate the role of the gut microbiota on CAR T cell therapy and suggest that modulation of the gut microbiota using vancomycin may improve outcomes after CAR T cell therapy across tumor types.

OsPUB9 Gene Edited by CRISPR/Cas9 Enhanced Resistance to Bacterial Leaf Blight in Rice (Oryza sativa L.).

Ubiquitination plays a crucial role in regulating signal pathways during the post-translation stage of protein synthesis in response to various environmental stresses. E3 ubiquitin ligase has been discovered to ultimately control various intracellular activities by imparting specificity to proteins to be degraded. This study was conducted to confirm biological and genetic functions of the U-box type E3 ubiquitin ligase (PUB) gene against biotic stress in rice (Oryza sativa L.). OsPUB9 gene-specific sgRNA were designed and transformants were developed through Agrobacterium-mediated transformation. Deep sequencing using callus was performed to confirm the mutation type of T0 plants, and a total of three steps were performed to select null individuals without T-DNA insertion. In the case of the OsPUB9 gene-edited line, a one bp insertion was generated by gene editing, and it was confirmed that early stop codon and multiple open reading frame (ORF) sites were created by inserting thymine. It is presumed that ubiquitination function also changed according to the change in protein structure of U-box E3 ubiquitin ligase. The OsPUB9 gene-edited null lines were inoculated with bacterial leaf blight, and finally confirmed to have a resistance phenotype similar to Jinbaek, a bacterial blight-resistant cultivar. Therefore, it is assumed that the amino acid sequence derived from the OsPUB9 gene is greatly changed, resulting in a loss of the original protein functions related to biological mechanisms. Comprehensively, it was confirmed that resistance to bacterial leaf blight stress was enhanced when a mutation occurred at a specific site of the OsPUB9 gene.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Enhanced selectivity and recovery of phosphate and nitrate ions onto coffee ground waste biochars via co-precipitation of Mg/Al layered double hydroxides: A potential slow-release fertilizer.

In this study, the feasibility of Mg/Al layered double hydroxides (LDH) functionalized coffee ground waste biochars (LDHMgAl@CWGB) as a potential adsorbent to selectively recover phosphate (PO43-) and nitrate (NO3-) ions in aqueous phases and their consecutive uses as a slow-release fertilizer for stimulating the plant growth were identified. The higher adsorption capacity of PO43- and NO3- ions by LDHMgAl@CWGB (PO43- = 6.98 mgP/g, NO3- = 2.82 mgN/g) compared with pristine coffee ground waste biochars (CWGB; PO43- = 0.19 mgP/g, NO3- = 0.32 mgN/g) was mainly due to the incorporation of Mg/Al mixed oxides and Cl contents. Chemisorption and intra-particle mainly controlled the adsorptive recovery of PO43- and NO3- ions by CWGB and LDHMgAl@CWGB in aqueous phases and their adsorption toward CWGB and LDHMgAl@CWGB proceeded endothermically and spontaneously. The changes in the major adsorption mechanisms of PO43- and NO3- ions from ligand exchange (CWGB) to electrostatic surface complexation and anion-exchange (LDHMgAl@CWGB) supported the conclusion that the alternation of the surface features through Mg/Al LDH functionalization might improve selectivity and adsorption capacity of PO43- and NO3- ions onto CWGB under the co-existence of Cl-, SO42-, and HCO3- ions. Since PO43-- and NO3--loaded LDHMgAl@CWGB exhibited much higher seed germination, root and shoot growth rates of garden cress seeds (Lepidium sativum L) than other liquid and solid matrices, including 5 mgP/L PO43- and 5 mgN/L NO3-, 10 mgP/L PO43- and 10 mgN/L NO3-, and LDHMgAl@CWGB, it can be postulated that PO43-- and NO3--loaded LDHMgAl@CWGB could be practically applicable to the agricultural field as a slow-release fertilizer to facilitate the seed germination, root and shoot growth of the plants.

Genetic Analysis Based on CRISPR/Cas9 Technology in Plants.

Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...].

Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice.

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T2OsPUB7 gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.

Intravenous to Oral Transition of Amiodarone (IOTA): Effect of Various Durations of Overlap on Atrial Fibrillation Recurrence After Cardiothoracic Surgery.

ABSTRACT: The use of amiodarone for postoperative atrial fibrillation (AF) is widespread; however, there is a paucity of data on the optimal duration of overlap when transitioning from intravenous (IV) to oral amiodarone. The objective of this study was to evaluate the safety and efficacy of varying durations of overlap when amiodarone IV infusion is transitioned to oral administration in cardiothoracic surgery patients. This retrospective, observational, single-center study included cardiothoracic surgery patients who were initiated on IV amiodarone for supraventricular arrhythmia and subsequently transitioned to oral amiodarone. The primary outcome was AF recurrence within 24 hours after IV amiodarone discontinuation. Safety outcomes include occurrence of bradycardia or hypotension while on amiodarone. A total of 184 patients were included for analysis. AF recurrence occurred in 24.5% of patients (n = 45). No significant association was found between various overlap durations and AF recurrence (odds ratio (OR) 1.00, 95% CI 1.00-1.01, P = 0.9). In addition, no significant association was found between duration of overlap and rates of bradycardia (OR 1.00, 95% confidence interval (CI) 0.99-1.00, P = 0.08) or hypotension (OR 1.00, 95% CI 0.99-1.00, P = 0.21), which occurred in 35.9% and 47.3% of patients, respectively. Our study suggests following conversion to normal sinus rhythm; cardiothoracic surgery patients can effectively and safely be transitioned from IV to oral amiodarone without the need for specific overlap duration or transition strategy.

Transcriptional Comparison of Genes Associated with Photosynthesis, Photorespiration, and Photo-Assimilate Allocation and Metabolic Profiling of Rice Species.

The ever-increasing human population alongside environmental deterioration has presented a pressing demand for increased food production per unit area. As a consequence, considerable research effort is currently being expended in assessing approaches to enhance crop yields. One such approach is to harness the allelic variation lost in domestication. This is of particular importance since crop wild relatives often exhibit better tolerance to abiotic stresses. Here, we wanted to address the question as to why wild rice species have decreased grain production despite being characterized by enhanced rates of photosynthesis. In order to do so, we selected ten rice species on the basis of the presence of genome information, life span, the prominence of distribution, and habitat type and evaluated the expression of genes in photosynthesis, photorespiration, sucrose and starch synthesis, sucrose transport, and primary and secondary cell walls. We additionally measured the levels of a range of primary metabolites via gas chromatography-mass spectrometry. The results revealed that the wild rice species exhibited not only higher photosynthesis but also superior CO2 recovery by photorespiration; showed greater production of photosynthates such as soluble sugars and starch and quick transportation to the sink organs with a possibility of transporting forms such as RFOs, revealing the preferential consumption of soluble sugars to develop both primary and secondary cell walls; and, finally, displayed high glutamine/glutamic acid ratios, indicating that they likely exhibited high N-use efficiency. The findings from the current study thus identify directions for future rice improvement through breeding.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Design of Neuraminidase-Targeted Imaging and Therapeutic Agents for the Diagnosis and Treatment of Influenza Virus Infections.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir-99mTc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.

Changes in adsorption mechanisms of radioactive barium, cobalt, and strontium ions using spent coffee waste biochars via alkaline chemical activation: Enrichment effects of O-containing functional groups.

The single adsorption of radioactive barium (Ba(II)), cobalt (Co(II)), and strontium (Sr(II)) ions using pristine (SCWB-P) and chemically activated spent coffee waste biochars with NaOH (SCWB-A) were thoroughly explored in order to provide deeper insights into the changes in their adsorption mechanisms through alkaline chemical activation. The greater removal efficiencies of SCWB-A (76.6-97.3%) than SCWB-P (45.6-75.2%) and the consistency between the adsorptive removal patterns (Ba(II) > Sr(II) > Co(II)) and oxygen bond dissociation enthalpies (BaO (562 kJ/mol) > SrO (426 kJ/mol) > CoO (397 kJ/mol)) of radioactive species supported the assumption that the adsorption removal of radioactive species with spent coffee waste biochars highly depended on the abundances of O-containing functional groups. The calculated R2 values of the pseudo-first-order (SCWB-P = 0.998-0.999; SCWB-A = 0.850-0.921) and pseudo-second-order kinetic models (SCWB-P = 0.988-0.998; SCWB-A = 0.935-0.966) are evident that the physisorption mainly controlled the adsorption of radioactive species toward SCWB-P and the chemisorption played a crucial role in their adsorptive removal with SCWB-A. From the calculated intra-particle diffusion, isotherm, thermodynamic parameters, it can be concluded that the intra-particle diffusion and monolayer adsorption primarily governed the adsorption of radioactive species using SCWB-P and SCWB-A, and their adsorption processes occurred spontaneously and endothermically. The dominant adsorption mechanism of spent coffee waste biochars was changed from physisorption (ΔH° of SCWB-P = 21.6-29.8 kJ/mol) to chemisorption (ΔH° of SCWB-A = 42.4-81.3 kJ/mol) through alkaline chemical activation. The distinctive M-OH peak in the O1s XPS spectra of SCWB-A directly corresponding to the decrease in the abundances of O-containing functional groups confirms again that the enrichment of O-containing functional groups markedly facilitated the adsorption removal of radioactive species by chemisorption occurred at the inner and outer surfaces of spent coffee waste biochars.

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

KEY MESSAGE: The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T0 plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.

Molecular and Functional Analysis of U-box E3 Ubiquitin Ligase Gene Family in Rice (Oryzasativa).

Proteins encoded by U-box type ubiquitin ligase (PUB) genes in rice are known to play an important role in plant responses to abiotic and biotic stresses. Functional analysis has revealed a detailed molecular mechanism involving PUB proteins in relation to abiotic and biotic stresses. In this study, characteristics of 77 OsPUB genes in rice were identified. Systematic and comprehensive analyses of the OsPUB gene family were then performed, including analysis of conserved domains, phylogenetic relationships, gene structure, chromosome location, cis-acting elements, and expression patterns. Through transcriptome analysis, we confirmed that 16 OsPUB genes show similar expression patterns in drought stress and blast infection response pathways. Numerous cis-acting elements were found in promoter sequences of 16 OsPUB genes, indicating that the OsPUB genes might be involved in complex regulatory networks to control hormones, stress responses, and cellular development. We performed qRT-PCR on 16 OsPUB genes under drought stress and blast infection to further identify the reliability of transcriptome and cis-element analysis data. It was confirmed that the expression pattern was similar to RNA-sequencing analysis results. The transcription of OsPUB under various stress conditions indicates that the PUB gene might have various functions in the responses of rice to abiotic and biotic stresses. Taken together, these results indicate that the genome-wide analysis of OsPUB genes can provide a solid basis for the functional analysis of U-box E3 ubiquitin ligase genes. The molecular information of the U-box E3 ubiquitin ligase gene family in rice, including gene expression patterns and cis-acting regulatory elements, could be useful for future crop breeding programs by genome editing.

Real-time biomonitoring of oxygen uptake rate and biochemical oxygen demand using a novel optical biogas respirometric system.

In response to the ever-increasing need for monitoring-based process control of wastewater treatment plants, an online applicable respirometer shows great promise for real-time measurement of oxygen uptake rate (OUR) and biochemical oxygen demand (BOD) measurements as a surrogate of the biodegradability of wastewater. Here, we have developed a photosensor-assisted real-time respirometric system equipped with bubble counting sensors for accurate measurement of microbial oxygen consumption in a bottle. This system can measure OUR and BOD in a bottle equipped with a tube containing NaOH solution to absorb carbon dioxide and supplied with continuous atmospheric oxygen to the bottle, which reliably supplies non-limiting dissolved oxygen (DO) for aerobic biodegradation even at high organic loads. These technical improvements allow a sensitive and rapid analytical tool offering real-time profiles of oxygen uptake rate as well as BOD measurements with an extended measurable range (0-420 mg O2/L), enabling significant reduction or elimination of dilution steps. The respirometric system was used to elucidate the biodegradable kinetics of domestic and swine wastewaters as a function of the type and concentration of organic matters, depending on source characteristics including rapidly or slowly oxidizable organic substances by bacteria. Compared with conventional and manometric BOD methods, our method is reliable and accurate.

The 24-Hour Intensivists Staffing Model Improves the Outcome for Nighttime Admitted Patients: A Matched Historical Control Study.

INTRODUCTION: We previously showed that a "10-hour daytime on-site" and "nighttime (NT) on-call" staffing strategy was associated with higher mortality for intensive care unit (ICU) patients admitted during NT than it was for patients admitted during office hours (OH). In here, we evaluated the clinical effects of a 24-hour intensivist staffing model. METHODS: We formed an intervention group of 3034 consecutive ICU patients hospitalized from January 2013 to December 2015, and a control group of 2891 patients from our previous study (2009-2011). We applied propensity score matching (PSM) for whole and subgroup analyses adjusting for confounding factors. We compared clinical outcomes of patients under the 2 staffing models using multivariate logistic regression and survival analyses. RESULTS: After PSM, we balanced the clinical data between the complete cohorts and the subgroups. Comparison of ICU survivals between the intervention and control cohorts yielded no significant differences. However, the intervention was significantly associated with a higher ICU survival in the NT (5:30 pm-07:30 am) admission patients (P = .049) than in those admitted during OH (07:30 am to 5:30 pm; P = .456). Additionally, the intervention shortened the LOSHOS (P = .001) and/or LOSICU (P < .001), reduced the hospital (P = .672) and/or ICU (P = .004) expenses, and resulted in earlier mechanical ventilation extubation (P = .442) as compared to the same variables in the control group, especially for NT admissions. CONCLUSIONS: The 24-hour intensivists staffing could significantly improve ICU outcomes, especially for NT-admission patients in high-acuity, high-volume ICUs with frequent NT admissions.

Enhanced mechanical deep dewatering of dewatered sludge by a thermal hydrolysis pre-treatment: Effects of temperature and retention time.

This study investigated effects of the thermal hydrolysis pre-treatment on mechanical deep dewaterability of dewatered sludge to extend understanding of dewatering characteristics of thermally hydrolyzed sludge. Floc sizes of dewatered sludge were gradually reduced during the thermal hydrolysis pre-treatment at 170 °C and 185 °C with increasing retention time whereas longer retention time (>60 min) increased floc sizes of thermally hydrolyzed sludges at 200 °C due to formation of undesired refractory organic materials (ROMs), which might hinder the disintegration of dewatered sludge flocs. Similar trends were found for thermal hydrolytic solubilization of dewatered sludge. This demonstrated that the efficiency of the thermal hydrolysis pre-treatment at a higher temperature (200 °C) with longer retention time (≥60 min) could be strongly influenced by the formation of ROMs associated with changes of solid fractions and some free amino acids (i.e., ß-aminobutyric acid, 4-hydroxyproline, and cysteine). Since the trade-off between the degradation of dewatered sludge and the formation of ROMs determined mechanical deep dewaterability of thermally hydrolyzed sludge, the lowest residual weight and moisture content were observed for thermally hydrolyzed sludges at 200 °C with retention time range of 60 min (residual weight = 0.165; moisture content = 55.38%) to 90 min (residual weight = 0.160; moisture content = 59.87%). These observations were intimately correlated to variations of extracellular polymeric substances during the thermal hydrolysis pre-treatment, but not in accordance with the change pattern of capillary suction time (CST) values. This is evident that the CST value was inadequate to estimate mechanical deep dewaterability of thermally hydrolyzed sludge.

Genome-wide transcriptional response of papain-like cysteine protease-mediated resistance against Xanthomonas oryzae pv. oryzae in rice.

KEY MESSAGE: Transgenic rice overexpressing PLCP attenuated the virulence of Xanthomonas oryzae pv. oryzae through extensive activation of transduction signal and transcription activities that orchestrate downstream responses including the biosynthesis of secondary metabolites and up-regulation of several pathogenesis-related proteins. High-throughput transcriptome investigations of plant immunity highlight the complexity of gene networks leading to incompatible interaction with the pathogen. Accumulating findings implicate papain-like cysteine proteases (PLCPs) as a central hub in plant defense. While diverse roles of PLCPs in different pathosystems have become more evident, information on gene networks and signaling pathways necessary to orchestrate downstream responses are lacking. To understand the biological significance of cysteine protease against Xanthomonas oryzae pv. oryzae, PLCP overexpression and knockout rice lines were generated. The pathogenicity test revealed the attenuation of Xanthomonas oryzae pv. oryzae race K3a virulence in transgenic lines which is ascribed to high hydrogen peroxide and free salicylic acid accumulation. Next-generation sequencing of RNA from transgenic and wild-type plants identified 1597 combined differentially expressed genes, 1269 of which were exclusively regulated in the transgenic libraries. It was found that PLCP aids rice to circumvent infection through the extensive activation of transduction signal and transcription factors that orchestrate downstream responses, including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites.