CTLA-4 tail fusion enhances CAR-T antitumor immunity.

Chimeric antigen receptor (CAR)-T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail, we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex or triplex CTLA-4 cytoplasmic tails (CCTs) fused to the C terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of proinflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior antitumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

Neighboring macrophage-induced alteration in the phenotype of colorectal cancer cells in the tumor budding area.

BACKGROUND: A higher number of tumor buds in the invasive front of colorectal cancer (CRC) specimens has been shown to contribute to a poor prognosis in CRC patients. Because macrophages (Mφs) have been demonstrated to alter the phenotype of cancer cells, we hypothesized that the phenotype of CRC cells in the tumor budding (TB) area might be changed by the interaction between CRC cells and Mφs. METHODS: We assessed the expression of topoisomerase 1 in CRC cells to estimate the acquisition of chemoresistance in CRC. To demonstrate the tumor-stromal interaction between CRC cells and Mφs, we assessed two histological findings, the number of Mφs per single CRC cell and the proximity between CRC cells and Mφs by histological spatial analysis using HALO software. RESULTS: The expression levels of topoisomerase 1 in CRC cells were decreased in deeper areas, especially in the TB area, compared to the surface area. Our histological spatial analysis revealed that 2.6 Mφs located within 60 µm of a single CRC cell were required to alter the phenotype of the CRC cell. Double-immunofluorescence staining revealed that higher Mφs were positive for interleukin-6 (IL-6) in the TB area and that AE1/AE3-positive CRC cells were also positive for phospho-STAT3 (pSTAT3) in the TB area; thus, the IL-6 receptor (IL-6R)/STAT3 signaling pathway in CRC cells was upregulated by IL-6 derived from neighboring Mφs. CONCLUSION: IL-6 secreted from the neighboring Mφs would alter the phenotype of CRC cells via IL-6R/STAT3 signaling pathway.

Examining amyloid reduction as a surrogate endpoint through latent class analysis using clinical trial data for dominantly inherited Alzheimer's disease.

INTRODUCTION: Increasing evidence suggests that amyloid reduction could serve as a plausible surrogate endpoint for clinical and cognitive efficacy. The double-blind phase 3 DIAN-TU-001 trial tested clinical and cognitive declines with increasing doses of solanezumab or gantenerumab. METHODS: We used latent class (LC) analysis on data from the Dominantly Inherited Alzheimer Network Trials Unit 001 trial to test amyloid positron emission tomography (PET) reduction as a potential surrogate biomarker. RESULTS: LC analysis categorized participants into three classes: amyloid no change, amyloid reduction, and amyloid growth, based on longitudinal amyloid Pittsburgh compound B PET standardized uptake value ratio data. The amyloid-no-change class was at an earlier disease stage for amyloid amounts and dementia. Despite similar baseline characteristics, the amyloid-reduction class exhibited reductions in the annual decline rates compared to the amyloid-growth class across multiple biomarker, clinical, and cognitive outcomes. DISCUSSION: LC analysis indicates that amyloid reduction is associated with improved clinical outcomes and supports its use as a surrogate biomarker in clinical trials. HIGHLIGHTS: We used latent class (LC) analysis to test amyloid reduction as a surrogate biomarker. Despite similar baseline characteristics, the amyloid-reduction class exhibited remarkably better outcomes compared to the amyloid-growth class across multiple measures. LC analysis proves valuable in testing amyloid reduction as a surrogate biomarker in clinical trials lacking significant treatment effects.

Amyloid-Related Imaging Abnormalities in the DIAN-TU-001 Trial of Gantenerumab and Solanezumab: Lessons from a Trial in Dominantly Inherited Alzheimer Disease.

OBJECTIVE: To determine the characteristics of participants with amyloid-related imaging abnormalities (ARIA) in a trial of gantenerumab or solanezumab in dominantly inherited Alzheimer disease (DIAD). METHODS: 142 DIAD mutation carriers received either gantenerumab SC (n = 52), solanezumab IV (n = 50), or placebo (n = 40). Participants underwent assessments with the Clinical Dementia Rating® (CDR®), neuropsychological testing, CSF biomarkers, ß-amyloid positron emission tomography (PET), and magnetic resonance imaging (MRI) to monitor ARIA. Cross-sectional and longitudinal analyses evaluated potential ARIA-related risk factors. RESULTS: Eleven participants developed ARIA-E, including 3 with mild symptoms. No ARIA-E was reported under solanezumab while gantenerumab was associated with ARIA-E compared to placebo (odds ratio [OR] = 9.1, confidence interval [CI][1.2, 412.3]; p = 0.021). Under gantenerumab, APOE-ɛ4 carriers were more likely to develop ARIA-E (OR = 5.0, CI[1.0, 30.4]; p = 0.055), as were individuals with microhemorrhage at baseline (OR = 13.7, CI[1.2, 163.2]; p = 0.039). No ARIA-E was observed at the initial 225 mg/month gantenerumab dose, and most cases were observed at doses >675 mg. At first ARIA-E occurrence, all ARIA-E participants were amyloid-PET+, 60% were CDR >0, 60% were past their estimated year to symptom onset, and 60% had also incident ARIA-H. Most ARIA-E radiologically resolved after dose adjustment and developing ARIA-E did not significantly increase odds of trial discontinuation. ARIA-E was more frequently observed in the occipital lobe (90%). ARIA-E severity was associated with age at time of ARIA-E. INTERPRETATION: In DIAD, solanezumab was not associated with ARIA. Gantenerumab dose over 225 mg increased ARIA-E risk, with additional risk for individuals APOE-ɛ4(+) or with microhemorrhage. ARIA-E was reversible on MRI in most cases, generally asymptomatic, without additional risk for trial discontinuation. ANN NEUROL 2022;92:729-744.

Accelerated longitudinal changes and ordering of Alzheimer disease biomarkers across the adult lifespan.

The temporal evolutions and relative orderings of Alzheimer disease biomarkers, including CSF amyloid-ß42 (Aß42), Aß40, total tau (Tau) and phosphorylated tau181 (pTau181), standardized uptake value ratio (SUVR) from the molecular imaging of cerebral fibrillar amyloid-ß with PET using the 11C-Pittsburgh Compound-B (PiB), MRI-based hippocampal volume and cortical thickness and cognition have been hypothesized but not yet fully tested with longitudinal data for all major biomarker modalities among cognitively normal individuals across the adult lifespan starting from 18 years. By leveraging a large harmonized database from 8 biomarker studies with longitudinal data from 2609 participants in cognition, 873 in MRI biomarkers, 519 in PET PiB imaging and 475 in CSF biomarkers for a median follow-up of 5-6 years, we estimated the longitudinal trajectories of all major Alzheimer disease biomarkers as functions of baseline age that spanned from 18 to 103 years, located the baseline age window at which the longitudinal rates of change accelerated and further examined possible modifying effects of apolipoprotein E (APOE) genotype. We observed that participants 18-45 years at baseline exhibited learning effects on cognition and unexpected directions of change on CSF and PiB biomarkers. The earliest acceleration of longitudinal change occurred for CSF Aß42 and Aß42/Aß40 ratio (with an increase) and for Tau, and pTau181 (with a decrease) at the next baseline age interval of 45-50 years, followed by an accelerated increase for PiB SUVR at the baseline age of 50-55 years and an accelerated decrease for hippocampal volume at the baseline age of 55-60 years and finally by an accelerated decline for cortical thickness and cognition at the baseline age of 65-70 years. Another acceleration in the rate of change occurred at the baseline age of 65-70 years for Aß42/Aß40 ratio, Tau, pTau181, PiB SUVR and hippocampal volume. Accelerated declines in hippocampal volume and cognition continued after 70 years. For participants 18-45 years at baseline, significant increases in Aß42 and Aß42/Aß40 ratio and decreases in PiB SUVR occurred in APOE ɛ4 non-carriers but not carriers. After age 45 years, APOE ɛ4 carriers had greater magnitudes than non-carriers in the rates of change for all CSF biomarkers, PiB SUVR and cognition. Our results characterize the temporal evolutions and relative orderings of Alzheimer disease biomarkers across the adult lifespan and the modification effect of APOE ɛ4. These findings may better inform the design of prevention trials on Alzheimer disease.

Cross-sectional and longitudinal comparisons of biomarkers and cognition among asymptomatic middle-aged individuals with a parental history of either autosomal dominant or late-onset Alzheimer's disease.

BACKGROUND: Comparisons of late-onset Alzheimer's disease (LOAD) and autosomal dominant AD (ADAD) are confounded by age. METHODS: We compared biomarkers from cerebrospinal fluid (CSF), magnetic resonance imaging, and amyloid imaging with Pittsburgh Compound-B (PiB) across four groups of 387 cognitively normal participants, 42 to 65 years of age, in the Dominantly Inherited Alzheimer Network (DIAN) and the Adult Children Study (ACS) of LOAD: DIAN mutation carriers (MCs) and non-carriers (NON-MCs), and ACS participants with a positive (FH+) and negative (FH-) family history of LOAD. RESULTS: At baseline, MCs had the lowest age-adjusted level of CSF Aß42 and the highest levels of total and phosphorylated tau-181, and PiB uptake. Longitudinally, MC had similar increase in PiB uptake to FH+, but drastically faster decline in hippocampal volume than others, and was the only group showing cognitive decline. DISCUSSION: Preclinical ADAD and LOAD share many biomarker signatures, but cross-sectional and longitudinal differences may exist.

Pattern and implications of neurological examination findings in autosomal dominant Alzheimer disease.

INTRODUCTION: As knowledge about neurological examination findings in autosomal dominant Alzheimer disease (ADAD) is incomplete, we aimed to determine the frequency and significance of neurological examination findings in ADAD. METHODS: Frequencies of neurological examination findings were compared between symptomatic mutation carriers and non mutation carriers from the Dominantly Inherited Alzheimer Network (DIAN) to define AD neurological examination findings. AD neurological examination findings were analyzed regarding frequency, association with and predictive value regarding cognitive decline, and association with brain atrophy in symptomatic mutation carriers. RESULTS: AD neurological examination findings included abnormal deep tendon reflexes, gait disturbance, pathological cranial nerve examination findings, tremor, abnormal finger to nose and heel to shin testing, and compromised motor strength. The frequency of AD neurological examination findings was 65.1%. Cross-sectionally, mutation carriers with AD neurological examination findings showed a more than two-fold faster cognitive decline and had greater parieto-temporal atrophy, including hippocampal atrophy. Longitudinally, AD neurological examination findings predicted a significantly greater decline over time. DISCUSSION: ADAD features a distinct pattern of neurological examination findings that is useful to estimate prognosis and may inform clinical care and therapeutic trial designs.

Pathogenesis of follicular thymic hyperplasia associated with rheumatoid arthritis.

Lymphoproliferative disorders may occur in patients with rheumatoid arthritis (RA) who are treated with methotrexate. However, follicular thymic hyperplasia (FTH) associated with RA (FTH-RA) is generally not considered a lymphoproliferative disorder. To investigate the pathogenesis of FTH-RA, we examined 12 cases of FTH involving thymic enlargement, four of FTH involving RA and eight of FTH involving myasthenia gravis (MG). Increased numbers and larger germinal center (GC) size were observed in FTH-RA group. The percentage of distorted GCs was 13.3% in FTH-RA group and 3.25% in FTH associated with MG (FTH-MG) group. A greater meshwork of follicular dendritic cells was observed in the GCs of FTH-RA group. Positive indices of CD27+ cells and PD-1+ cells per GC in FTH-RA group were significantly higher than those in FTH-MG group, though positive indices of CD68+ cells and CD163+ cells were similar. Myoid cell proliferation, as evaluated by α-SMA, tenascin-C, and l-caldesmon expression, was significantly increased in the FTH-RA group compared with the FTH-MG group. These results suggest that FTH should be considered in patients with RA treated with methotrexate. The pathogenesis of FTH-RA includes GC expansion and increased numbers of memory B cells, follicular helper T cells, and myoid cells, indicating humoral immunity activation.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

[A Case of Suspected Non-Occlusive Mesenteric Ischemia following Neoadjuvant Chemotherapy for Esophageal Cancer with an Extremely Poor Prognosis].

Non-occlusive mesenteric ischemia(NOMI)is defined as intestinal ischemia or necrosis with patency of the mesenteric arteries. Here, we report a case of suspected NOMI following neoadjuvant chemotherapy for esophageal cancer with an extremely poor prognosis. A 79-year-old man complained of weight loss and vomiting. Esophagogastroduodenoscopy revealed a tumor extending from the lower intrathoracic esophagus to the gastric cardia. He was diagnosed with esophageal cancer(small cell neuroendocrine carcinoma, T3(AD)N0M0, cStage Ⅱ)accordingly. He received cisplatin and etoposide as neoadjuvant chemotherapy. Tube feeding was initiated due to tumor stenosis. His weight increased rapidly by more than 8 kg on the second day of treatment. He did not display any signs of heart failure, and so continued chemotherapy in conjunction with diuretics. Upon completion of chemotherapy, his continued use of diuretics gradually reduced his weight. On day 7, the patient complained of nausea and experienced a decrease in blood pressure. Bicarbonate Ringer's solution was administered intravenously, but the patient lost consciousness after 3 hours. Plain computed tomography revealed massive gas collections in the portal vein, tumor wall, stomach, and ascending colon. NOMI was strongly suspected. His condition continued to deteriorate, until his demise several hours later. Here, we present the above-mentioned case and discuss the relevant literature.

Efficacy and Cost-effectiveness of Promotion Methods to Recruit Participants to an Online Screening Registry for Alzheimer Disease Prevention Trials: Observational Study.

BACKGROUND: Web-based screening may be suitable for identifying individuals with presymptomatic latent diseases for recruitment to clinical studies, as such people do not often visit hospitals in the presymptomatic stage. The promotion of such online screening studies is critical to their success, although it remains uncertain how the effectiveness of such promotion can differ, depending on the different promotion methods, domains of interest, or countries of implementation. OBJECTIVE: The Japanese Trial-Ready Cohort (J-TRC) web study is our ongoing online screening registry to identify individuals with presymptomatic Alzheimer disease (AD), aimed at facilitating the clinical trials for AD prevention. Within the first 9 months of its 2019 launch, the J-TRC web study recruited thousands of online participants via multiple methods of promotion, including press releases, newspaper advertisements, web advertisements, or direct email invitations. Here, we aimed to quantitatively evaluate efficacy and cost-effectiveness of each of these multimodal promotion methods. METHODS: We applied the vector-autoregression model to assess the degree of contribution of each type of promotion to the following target metrics: number of daily visitors to the J-TRC website, number of daily registrants to the J-TRC web study, daily rate of registration among visitors, daily rate of eligible participants among registrants, and median age of daily registrants. The average cost-effectiveness for each promotion method was also calculated using the total cost and the coefficients in the vector-autoregression model. RESULTS: During the first 9 months of the reviewed period from October 31, 2019 to June 17, 2020, there were 48,334 website visitors and 4429 registrations (9.16% of 48,334 visitors), of which 3081 (69.56%) were eligible registrations. Initial press release reports and newspaper advertisements had a marked effect on increasing the number of daily visitors and daily registrants. Web advertisements significantly contributed to the increase in daily visitors (P<.001) but not to the daily registrants, and it also lowered the rate of registrations and the median age of daily registrants. Website visitors from the direct email invitation sent to other cognitive registries seem to have registered with the highest reliability. The calculated average cost-effectiveness for the initial press release was US $24.60 per visitor and US $96.10 per registrant, while the calculated average cost-effectiveness for the newspaper advertisements was US $28.60 per visitor and US $227.90 per registrant. CONCLUSIONS: Our multivariate time-series analysis showed that each promotion method had different features in their effect of recruiting participants to the J-TRC web study. Under the advertisement condition settings thus far, newspaper advertisements and initial press releases were the most effective promotion methods, with fair cost-effectiveness that was equivalent to earlier online studies. These results can provide important suggestions for future promotions for the recruitment of presymptomatic participants to AD clinical trials in Japan.

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor.

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu2+ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, tp , for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of tp in the presence of excess Cu2+ was around 75°C, indicating that Cu2+ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu2+ ⇌ U∙Cu2+ , where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol-1 in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu2+ complex itself.

Good prognosis for follicular lymphoma with estrogen receptor α-positive follicular dendritic cells.

Follicular lymphoma (FL) has a meshwork of follicular dendritic cells (FDCs). We previously demonstrated the presence of estrogen receptor alpha (ERα)+ CD23+ FDCs in grades 1-2 FL. The significance of FDCs as a prognostic factor in FL remains unknown. The current study aimed to compare clinicopathological features, including prognosis, between FL with and without ERα+ FDCs. This study evaluated the clinicopathological significance of ERα expression in 70 FL patients by immunostaining. The presence of ERα mRNA on FDCs from 5 FL patients was confirmed by CD21/ERα double staining (immunohistochemistry and in situ hybridization). We defined patients with frequent ERα expression as the ERαhigh group and those with infrequent ERα expression as the ERαlow group. Thirty-two patients were assigned to the ERαhigh group (45.7%), and 38 patients were assigned to the ERαlow group (54.3%). Both overall survival (OS) and progression-free survival (PFS) were significantly better in the ERαhigh group than in the ERαlow group (OS, log-rank, P = .0465; PFS, log-rank, P = .0336). Moreover, high ERα expression on FDCs was an independent prognostic factor for OS in both the univariate ([hazard ratio] HR, 0.163; P = .0260) and multivariate (HR, 0.050; P = .0188) analyses and for PFS in both the univariate (HR, 0.232; P = .0213) and multivariate (HR, 0.084; P = .0243) analyses. ERα mRNA expression was detected in CD21+ FDCs within the neoplastic follicles of FL patients. In conclusion, a neoplastic follicular microenvironment with ERα-positive FDCs might affect the grade and presence of the follicular pattern of FL and improve patient prognosis.

Chitinase system of Aeromonas salmonicida, and characterization of enzymes involved in chitin degradation.

The genes encoding chitin-degrading enzymes in Aeromonas salmonicida SWSY-1.411 were identified and cloned in Escherichia coli. The strain contained two glycoside hydrolase (GH) families 18 chitinases: AsChiA and AsChiB, two GH19 chitinases: AsChiC and AsChiD, and an auxiliary activities family 10 protein, lytic polysaccharide monooxygenase: AsLPMO10A. These enzymes were successfully expressed in E. coli and purified. AsChiB had the highest hydrolytic activity against insoluble chitin. AsChiD had the highest activity against water-soluble chitin. The peroxygenase activity of AsLPMO10A was lower compared to SmLPMO10A from Serratia marcescens. Synergism on powdered chitin degradation was observed when AsChiA and AsLPMO10A were combined with other chitinases of this strain. More than twice the increase of the synergistic effect was observed when powdered chitin was treated by a combination of AsLPMO10A with all chitinases. GH19 chitinases suppressed the hyphal growth of Trichoderma reesei.

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan.

In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira-positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re-isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome-to-genome distances and average nucleotide identity in silico using whole genome sequences and DNA-DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 T , = JCM 32515 T = CIP111620 T ), Leptospira ellinghausenii sp. nov., (type strain E18 T , = JCM 32516 T = CIP111618 T ) and Leptospira ryugenii sp. nov., (type strain YH101 T , = JCM 32518 T = CIP111617 T ) are proposed.

Preoperative transcatheter arterial embolization using a gelatin sponge for head and neck tumors.

Purpose: To evaluate the usefulness of preoperative transcatheter arterial embolization using a gelatin sponge for hypervascular head and neck tumors to reduce intraoperative blood loss (IBL).　　　　 Material and methods: Nineteen patients underwent preoperative transcatheter arterial embolization for hypervascular head and neck tumors using a gelatin sponge. The technical success rate, devascularization rate, IBL, and complications were evaluated. Angiography images obtained before and after preoperative embolization were compared in all patients, and the devascularization rate was assessed from the relative reduction rate of contrast agent volumes. Results: The technical success rate was 100%. The median devascularization rate was 95% (range, 75-100%). The median period between embolization and surgical resection was one day (range, 1-12 days). The median IBL was 122 ml (range, 0-3780 ml). Blood transfusions were required in three cases, and their IBL and devascularization rates were 850, 1959, and 3780 ml, and 75%, 90%, and 80%, respectively. There was a complication of cerebral embolism in one out of 19 cases (5%). Conclusions: Preoperative transcatheter arterial embolization using a gelatin sponge was feasible and may contribute to decreasing IBL.

Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 µm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M µm and 8.1 × 10-10 M µm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake.

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.

Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins.

The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIA(Glc) of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3' segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source.