Contributions of the anaplerotic reaction enzymes pyruvate carboxylase and phosphoenolpyruvate carboxylase to l-lysine production in Corynebacterium glutamicum.

Anaplerotic reactions catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) have important roles in the production of l-lysine to replenish oxaloacetic acid (OAA) in Corynebacterium glutamicum. However, the relative contributions of these enzymes to l-lysine production in C. glutamicum are not fully understood. In this study, using a parent strain (P) carrying a feedback inhibition-resistant aspartokinase with the T311I mutation, we constructed a PC gene-deleted mutant strain (PΔPC) and a PEPC gene-deleted mutant strain (PΔPEPC). Although the growth of both mutant strains was comparable to the growth of strain P, the maximum l-lysine production in strains PΔPC and PΔPEPC decreased by 14% and 49%, respectively, indicating that PEPC strongly contributed to OAA supply. l-Lysine production in strain PΔPC slightly decreased during the logarithmic phase, while production during the early stationary phase was comparable to production in strain P. By contrast, strain PΔPEPC produced l-lysine in an amount comparable to the production of strain P during the logarithmic phase; l-lysine production after the early stationary phase was completely stopped in strain PΔPEPC. These results indicate that OAA is supplied by both PC and PEPC during the logarithmic phase, while only PEPC can continuously supply OAA after the logarithmic phase.

Effectiveness and safety of granulocyte colony-stimulating factor priming regimen for acute myeloid leukemia: A systematic review and meta-analysis of the Clinical Practice Guideline for the use of G-CSF 2022 from the Japan Society of Clinical Oncology.

BACKGROUND: The outcomes of relapsed or refractory acute myeloid leukemia (AML) remain poor. Although the concomitant use of granulocyte colony-stimulating factor (G-CSF) and anti-chemotherapeutic agents has been investigated to improve the antileukemic effect on AML, its usefulness remains controversial. This study aimed to investigate the effects of G-CSF priming as a remission induction therapy or salvage chemotherapy. METHODS: We performed a thorough literature search for studies related to the priming effect of G-CSF using PubMed, Ichushi-Web, and the Cochrane Library. A qualitative analysis of the pooled data was performed, and risk ratios (RRs) with confidence intervals (CIs) were calculated and summarized. RESULTS: Two reviewers independently extracted and accessed the 278 records identified during the initial screening, and 62 full-text articles were assessed for eligibility in second screening. Eleven studies were included in the qualitative analysis and 10 in the meta-analysis. A systematic review revealed that priming with G-CSF did not correlate with an improvement in response rate and overall survival (OS). The result of the meta-analysis revealed the tendency for lower relapse rate in the G-CSF priming groups without inter-study heterogeneity [RR, 0.91 (95% CI 0.82-1.01), p = 0.08; I2 = 4%, p = 0.35]. In specific populations, including patients with intermediate cytogenetic risk and those receiving high-dose cytarabine, the G-CSF priming regimen prolonged OS. CONCLUSIONS: G-CSF priming in combination with intensive remission induction treatment is not universally effective in patients with AML. Further studies are required to identify the patient cohort for which G-CSF priming is recommended.

Direct Species Identification in Positive Blood Culture Bottles From Patients With Hematologic Malignancies.

Background In patients with hematologic malignancies, faster species identification is particularly important in the management of bloodstream infection because of their immunocompromised and neutropenic status. In the present study, we analyzed direct species identification in patients with hematologic malignancies, and the factors that might influence the results of species identification. Methods We performed direct species identification using a Sepsityper® kit (Bruker Corporation, Billerica, Massachusetts, United States) and compared the results with a conventional method in patients with hematologic malignancies. Forty-five positive blood culture bottles containing single microorganisms from 37 patients were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). And patients' clinical data were compared between the groups with spectral scores at acceptable and unacceptable levels. Results Direct species identification correctly identified 42 of 45 isolates and three were misidentified. While 35 of 45 isolates showed a spectral score ≥1.7 (acceptable identification), 10 isolates had a spectral score <1.7 (unacceptable identification) including three misidentified isolates. The group with a spectral score ≥1.7 had significantly lower white blood cell (p<0.01), neutrophil (p<0.01), and platelet (p<0.01) counts in addition to more frequent central venous (CV) line insertion (p=0.01). Multivariate analysis revealed that pathogen type (gram-positive or negative) and CV line insertion were associated with spectral scores. Conclusion Direct species identification using the Sepsityper kit is an upcoming approach for blood culture bottles, which were flagged as positive even in patients with hematologic malignancies when the spectral score was ≥ 1.7. Our study also indicates that direct identification is more accurate in patients with CV lines, and may be less accurate when gram-positive bacteria are detected.

Effectiveness and safety of primary prophylaxis with G-CSF after induction therapy for acute myeloid leukemia: a systematic review and meta-analysis of the clinical practice guidelines for the use of G-CSF 2022 from the Japan society of clinical oncology.

Although granulocyte colony-stimulating factor (G-CSF) reduces the incidence, duration, and severity of neutropenia, its prophylactic use for acute myeloid leukemia (AML) remains controversial due to a theoretically increased risk of relapse. The present study investigated the effects of G-CSF as primary prophylaxis for AML with remission induction therapy. A detailed literature search for related studies was performed using PubMed, Ichushi-Web, and the Cochrane Library. Data were independently extracted and assessed by two reviewers. A qualitative analysis of pooled data was conducted, and the risk ratio with corresponding confidence intervals was calculated in the meta-analysis and summarized. Sixteen studies were included in the qualitative analysis, nine of which were examined in the meta-analysis. Although G-CSF significantly shortened the duration of neutropenia, primary prophylaxis with G-CSF did not correlate with infection-related mortality. Moreover, primary prophylaxis with G-CSF did not affect disease progression/recurrence, overall survival, or adverse events, such as musculoskeletal pain. However, evidence to support or discourage the use of G-CSF as primary prophylaxis for adult AML patients with induction therapy remains limited. Therefore, the use of G-CSF as primary prophylaxis can be considered for adult AML patients with remission induction therapy who are at a high risk of infectious complications.

Utility of allogeneic stem cell transplantation for adult Ph+ALL with complete molecular remission.

This study aimed to investigate the usefulness of allogeneic stem cell transplantation (allo-SCT) for Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) in the first complete remission (CR1) with complete molecular remission (CMR). We compared the outcomes between Ph+ALL patients who did or did not undergo allo-SCT in CR1. We included patients enrolled in the prospective clinical studies in the tyrosine kinase inhibitor era conducted by the Japan Adult Leukemia Study Group, who achieved CMR within 3 months. A total of 147 patients (allo-SCT: 101; non-SCT: 46) were eligible for this analysis. In the multivariate analyses, allo-SCT was significantly associated with both superior overall survival (OS) (adjusted hazard ratio (aHR): 0.54; 95% CI: 0.30-0.97; p = .04) and relapse-free survival (RFS) (aHR: 0.21; 95% CI: 0.12-0.38; p < .001). The 5-year adjusted OS and RFS were 73% and 70% in the allo-SCT cohort, whereas they were 50% and 20% in the non-SCT cohort. Despite the higher non-relapse mortality (aHR: 3.49; 95% CI: 1.17-10.4; p = .03), allo-SCT was significantly associated with a lower relapse rate (aHR: 0.10; 95% CI: 0.05-0.20; p < .001). In addition, allo-SCT was also associated with superior graft-versus-host disease-free, relapse-free survival (aHR: 0.43; 95% CI: 0.25-0.74; p = .002). Propensity score-matched analyses confirmed the results of the multivariate analyses. In patients who achieved CMR within 3 months, allo-SCT in CR1 had superior survival and lower relapse compared with the non-SCT cohort.

Clinical impact and characteristics of erythroid dysplasia in adult aplastic anaemia: Results from a multicentre registry.

Morphological dysplasia in haematopoietic cells, defined by a 10% threshold in each lineage, is one of the diagnostic criteria for myelodysplastic neoplasms. Dysplasia limited to the erythroid lineage has also been reported in some cases of aplastic anaemia (AA); however, its significance remains unclear. We herein examined the impact of erythroid dysplasia on immunosuppressive therapy responses and survival in AA patients. The present study included 100 eligible AA patients without ring sideroblasts. Among them, 32 had dysplasia in the erythroid lineage (AA with minimal dysplasia [mini-D]). No significant sex or age differences were observed between AA groups with and without erythroid dysplasia. In severe/very severe AA and non-severe AA patients, a response to anti-thymocyte globulin + ciclosporin within 12 months was observed in 80.0% and 60.0% of AA with mini-D and 42.9% and 90.0% of those without dysplasia, with no significant difference (p = 0.29 and p = 0.24 respectively). Overall survival and leukaemia-free survival did not significantly differ between the groups. Collectively, the present results indicate that the presence of erythroid dysplasia did not significantly affect clinical characteristics or outcomes in AA patients, suggesting that its presence in AA is acceptable. Therefore, erythroid dysplasia should not exclude an AA diagnosis.

Laboratory Evolution of Antimicrobial Resistance in Bacteria to Develop Rational Treatment Strategies.

Laboratory evolution studies, particularly with Escherichia coli, have yielded invaluable insights into the mechanisms of antimicrobial resistance (AMR). Recent investigations have illuminated that, with repetitive antibiotic exposures, bacterial populations will adapt and eventually become tolerant and resistant to the drugs. Through intensive analyses, these inquiries have unveiled instances of convergent evolution across diverse antibiotics, the pleiotropic effects of resistance mutations, and the role played by loss-of-function mutations in the evolutionary landscape. Moreover, a quantitative analysis of multidrug combinations has shed light on collateral sensitivity, revealing specific drug combinations capable of suppressing the acquisition of resistance. This review article introduces the methodologies employed in the laboratory evolution of AMR in bacteria and presents recent discoveries concerning AMR mechanisms derived from laboratory evolution. Additionally, the review outlines the application of laboratory evolution in endeavors to formulate rational treatment strategies.

Outcomes of transplant-eligible patients with myelodysplastic syndrome with excess blasts registered in an observational study: The JALSG-CS11-MDS-SCT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the sole curative therapy for myelodysplastic syndrome (MDS). However, whether bridging therapy (BRT) including azacitidine (AZA) and combination chemotherapy (CCT) prior to allo-SCT should be performed is unclear. We analyzed BRT and the outcomes of patients with myelodysplastic syndrome with excess blasts (MDS-EB) who were ≤ 70 years old at the time of registration for a prospective observational study to clarify the optimal allo-SCT strategy for high-risk MDS. A total of 371 patients were included in this study. Among 188 patients (50.7%) who were considered for allo-SCT, 141 underwent allo-SCT. Among the patients who underwent allo-SCT, 64 received AZA, 29 received CCT, and 26 underwent allo-SCT without BRT as the initial treatment. Multivariate analysis identified BRT as an independent factor influencing overall survival (AZA vs. without BRT, hazard ratio [HR] 3.33, P = 0.005; CCT vs. without BRT, HR 3.82, P = 0.003). In multivariate analysis, BRT was independently associated with progression-free survival (AZA vs. without BRT: HR, 2.23; P = 0.041; CCT vs. without BRT: HR, 2.94; P = 0.010). Transplant-eligible patients with MDS-EB should undergo allo-SCT when clinically acceptable, and upfront allo-SCT without BRT may be superior to AZA or CCT.

Recurrent Stroke with Rapid Development of Intracranial Artery Stenosis and Subsequent Successful Mechanical Thrombectomy in Essential Thrombocythemia.

Essential thrombocythemia is a myeloproliferative neoplasm. Ischemic stroke is frequently the first manifestation of essential thrombocythemia. We herein report a patient with JAK2V617 mutation-positive essential thrombocythemia who developed recurrent ischemic stroke with rapid development of intracranial artery stenosis and subsequently underwent successful mechanical thrombectomy. The high JAK2V617F allele burden in our patient (58.4%) may have affected the patient's condition. We discuss similar reports in the literature and the possible pathophysiologic mechanism of large artery involvement in these patients.

[Thoracic SMARCA4-deficient undifferentiated tumor resembling malignant lymphoma].

We present a case of thoracic SMARCA4-deficient undifferentiated tumor that needed to be differentiated from malignant lymphoma owing to multiple lymph node swelling and marrow involvement. A 52-year-old man developed multiple lymphadenopathies along with anorexia, general fatigue, fever, and sweating 2 months prior to admission. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) scan revealed a mass lesion on the right upper lung, generalized lymph node swelling, and bone metastasis, indicating the presence of suspicious lung cancer; therefore, he was referred to our hospital. Malignant lymphoma was suspected at the time of admission because of elevated levels of lactate dehydrogenase (11,977 U/l) and soluble interleukin 2 receptor (2,152 U/ml) as well as marrow infiltration of large abnormal cells. On day 11, the patient died from rapid respiratory failure. Histological and immunohistochemical features of the pleural effusion cell block led to the diagnosis of thoracic SMARCA4-deficient undifferentiated tumor. Thoracic SMARCA4-deficient undifferentiated tumor was recently introduced in the 2021 World Health Organization classification of lung tumors, with most patients being young adults with a history of heavy smoking and poor prognosis. Because of the multiple lymph node swelling and marrow involvement, this undifferentiated tumor should be distinguished from malignant lymphoma.

Analysis of the evolution of resistance to multiple antibiotics enables prediction of the Escherichia coli phenotype-based fitness landscape.

The fitness landscape represents the complex relationship between genotype or phenotype and fitness under a given environment, the structure of which allows the explanation and prediction of evolutionary trajectories. Although previous studies have constructed fitness landscapes by comprehensively studying the mutations in specific genes, the high dimensionality of genotypic changes prevents us from developing a fitness landscape capable of predicting evolution for the whole cell. Herein, we address this problem by inferring the phenotype-based fitness landscape for antibiotic resistance evolution by quantifying the multidimensional phenotypic changes, i.e., time-series data of resistance for eight different drugs. We show that different peaks of the landscape correspond to different drug resistance mechanisms, thus supporting the validity of the inferred phenotype-fitness landscape. We further discuss how inferred phenotype-fitness landscapes could contribute to the prediction and control of evolution. This approach bridges the gap between phenotypic/genotypic changes and fitness while contributing to a better understanding of drug resistance evolution.

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Prospective comparison of 5- and 7-day administration of azacitidine for myelodysplastic syndromes: a JALSG MDS212 trial.

The hypomethylating agent azacitidine (AZA) significantly extends overall survival (OS) in patients with higher risk myelodysplastic syndromes (MDS), when compared with other conventional care regimens, including supportive care and low-dose and intensive chemotherapy. However, the effects of 5- and 7-day treatment schedules of AZA (AZA-5 and AZA-7, respectively) on the OS of MDS patients had not been compared prospectively. We started a phase 3 trial comparing the effects of AZA-7 and AZA-5 on MDS patients with refractory anemia with excess blasts (RAEB) and RAEB in transformation (RAEB-T). However, this trial was prematurely terminated because of poor recruitment. Using all data, there was no significant difference in the OS of patients between AZA-7 (92 patients) and AZA-5 (95 patients), with the 2-year OS rates of AZA-7 and AZA-5 at 36.4% and 25.8%, respectively (P = 0.293). Adverse event profiles were similar between the two groups. Interestingly, data of the centrally diagnosed RAEB and RAEB-T cases showed that AZA-7 significantly prolonged the time to leukemia transformation compared with AZA-5 (P = 0.022), confirmed by multivariate analysis. Although this trial could not provide definite evidence, the results support the use of AZA-7 for RAEB and RAEB-T. (UMIN Clinical Trials Registry UMIN000009633).

Bio-Interface on Freestanding Nanosheet of Microelectromechanical System Optical Interferometric Immunosensor for Label-Free Attomolar Prostate Cancer Marker Detection.

Various biosensors that are based on microfabrication technology have been developed as point-of-care testing devices for disease screening. The Fabry-Pérot interferometric (FPI) surface-stress sensor was developed to improve detection sensitivity by performing label-free biomarker detection as a nanomechanical deflection of a freestanding membrane to adsorb the molecules. However, chemically functionalizing the freestanding nanosheet with excellent stress sensitivity for selective molecular detection may cause the surface chemical reaction to deteriorate the nanosheet quality. In this study, we developed a minimally invasive chemical functionalization technique to create a biosolid interface on the freestanding nanosheet of a microelectromechanical system optical interferometric surface-stress immunosensor. For receptor immobilization, glutaraldehyde cross-linking on the surface of the amino-functionalized parylene membrane reduced the shape variation of the freestanding nanosheet to 1/5-1/10 of the previous study and achieved a yield of 95%. In addition, the FPI surface-stress sensor demonstrated molecular selectivity and concentration dependence for prostate-specific antigen with a dynamic range of concentrations from 100 ag/mL to 1 µg/mL. In addition, the minimum limit of detection of the proposed sensor was 2,000,000 times lower than that of the conventional nanomechanical cantilevers.

Bendamustine-induced rash is associated with a favorable prognosis in patients with indolent B-cell lymphoma.

Bendamustine is now recognized as a key drug for indolent B-cell lymphoma (iBCL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Skin toxicity associated with bendamustine is one of the characteristic adverse effects. We retrospectively examined the relationship between bendamustine-associated drug rashes and disease prognosis of iBCL and MCL at our institution. Between January 2011 and August 2019, 65 patients (39 men and 26 women, median age 68, range 41-84 years) were treated with bendamustine alone (n=11, 120 mg/m2 on days 1 and 2) or a combination of rituximab and bendamustine (n=54, 90 mg/m2 on days 1 and 2). Of these patients, 47 had follicular lymphoma (FL), 10 had MCL and 8 had other iBCLs. Drug rash occurred in 27 (41.5%). Eight cases (29.6%) were grade 1, 5 (18.5%) were grade 2 and 14 (51.9%) were grade 3. The onset was in the first course in 17 (63.0%), 2nd course in 5 (18.5%), 3rd course in 2 (7.4%), 4th course in 1 (3.7%) and 5th course in 2 (7.4%). No treatment was administered in 1 case (3.7%), topical steroid was applied in 10 (37.0%), antiallergic drug was administered in 2 (7.4%), topical steroid and antiallergic drug were administered in 5 (18.5%), and oral and topical steroid and antiallergic drug were administered in 9 (33.3%). The 3-year progression-free survival (PFS) and overall survival (OS) in patients with rash development were 80.0% and 85.5%, respectively, and those in patients without development were 36.4% and 54.0%, respectively (p=0.009 and 0.02, respectively). By multivariate analysis, the development of rash was associated with a better PFS and a diagnosis of iBCL was associated with a better OS. This study revealed that bendamustine-induced rash is associated with a favorable prognosis among patients with iBCL.

Paravertebral extramedullary hematopoiesis in a case of myelodysplastic syndrome with ring sideroblasts and an SF3B1 mutation.

We present the case of a 56-year-old male patient with paravertebral extramedullary hematopoiesis (EMH) secondary to myelodysplastic syndrome with ring sideroblasts and multilineage dysplasia. In a routine health checkup over 5 years prior, he presented with asymptomatic mild anemia and a posterior mediastinal mass. Pathological and cytomorphological findings of the resected paravertebral mass were similar to those of his bone marrow specimen, and included cellularity with erythroid hyperplasia, multilineage dysplastic changes, and the presence of ring sideroblasts. A concordant SF3B1 mutation was detected in both bone marrow and paravertebral mass samples, suggesting that the EMH cells were derived from the bone marrow.

Acceleration of target production in co-culture by enhancing intermediate consumption through adaptive laboratory evolution.

Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)-E. coli (downstream: mevalonate to isoprenol) co-culture. An engineered mevalonate auxotroph strain was repeatedly sub-cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole-genome re-sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol-producing parental and evolved strains were respectively co-cultured with a mevalonate-producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain.

Adaptive Laboratory Evolution of Microorganisms: Methodology and Application for Bioproduction.

Adaptive laboratory evolution (ALE) is a useful experimental methodology for fundamental scientific research and industrial applications to create microbial cell factories. By using ALE, cells are adapted to the environment that researchers set based on their objectives through the serial transfer of cell populations in batch cultivations or continuous cultures and the fitness of the cells (i.e., cell growth) under such an environment increases. Then, omics analyses of the evolved mutants, including genome sequencing, transcriptome, proteome and metabolome analyses, are performed. It is expected that researchers can understand the evolutionary adaptation processes, and for industrial applications, researchers can create useful microorganisms that exhibit increased carbon source availability, stress tolerance, and production of target compounds based on omics analysis data. In this review article, the methodologies for ALE in microorganisms are introduced. Moreover, the application of ALE for the creation of useful microorganisms as cell factories has also been introduced.

[Treatment for myelodysplastic syndromes and future perspectives in the genomic era].

Myelodysplastic syndrome (MDS) is a group of clonal diseases caused by the accumulation of genetic mutations. The outcome of MDS extremely varies, with an overall survival ranging from just a few months to years. Therefore, accurate classification and prognostic scoring are essential. Patients with MDS are generally divided into two risk groups. For low-risk patients, the treatment goal is to improve ineffective hematopoiesis and quality of life. Meanwhile, in high-risk patients, treatment aims to extend survival and prevent progression to leukemia. To date, different guidelines recommend azacytidine, which is a hypomethylating agent, as the initial treatment. This is the only therapy associated with a significant survival in high-risk patients who are not eligible for hematopoietic stem cell transplantation. However, the response rate is only about 40%, and responses are mostly transient. Recent advancements in sequencing technologies have improved our understanding of the molecular pathogenesis of MDS by identifying somatic mutations in almost each patient with MDS. The high phenotypic and clinical heterogeneity of patients with MDS is primarily based on genetics. Due to a high degree of heterogeneity, the treatment policy for patients with MDS is still challenging. In this review, we will discuss the current treatment strategies for MDS in Japan, including future perspectives.