Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

Minimum 19-Year Clinical Results and Patient Satisfaction After Total Knee Arthroplasty.

BACKGROUND: Long-term (minimum 19-year) outcome data on clinical results and patient satisfaction after posterior-stabilized total knee arthroplasties (TKAs) are missing in the literature. The purpose of the study was to evaluate the clinical and radiographic results as well as patient satisfaction at a mean of 21.2 years after posterior-stabilized TKAs. METHODS: This study included 756 patients (1,350 knees) who had undergone TKAs. There were 96 men and 660 women (mean age, 58 years; range, 40 to 84). The mean follow-up was 21.2 years (range, 19 to 23). At each follow-up visit, the patients were assessed radiographically and clinically. Furthermore, patient satisfaction was determined. RESULTS: The Knee Society total, pain, function, and deformity scores were 42, 18, 33, and 5 points, respectively, at the final follow-up. The mean Western Ontario and McMaster Universities Arthritis Index score was 25 points at the final follow-up. With revision or aseptic loosening as the end point, the 23-year intimated survival for the implant was 96% (95% confidence interval, 91 to 100%). The overall patient satisfaction score at the final follow-up was 83.3 points (range, 81 to 86). Patient satisfaction scores with regard to pain, housework, recreation, and surgery were 84, 81, 82, and 86 points, respectively. CONCLUSIONS: The findings of the present, mean 21-year follow-up clinical study suggest excellent results with regard to the revision rates and survivorship of the posterior-stabilized total knee implants. However, consistent with the literature, we found that about 80% of patients expressed overall satisfaction with their primary TKAs. About 8% of patients were either somewhat or very dissatisfied with the procedure.

Long-term clinical results and patient satisfaction of a metaphyseal-engaging anatomic cementless femoral component in total hip arthroplasty.

PURPOSE: There is relatively little information on the long-term clinical results of patients aged < 50 years with a contemporary total hip arthroplasty (THA), although a high rate of revision is projected for this group. Therefore, the purpose of this study was to evaluate the long-term results (a minimum of 21 years) of a metaphyseal-engaging anatomic cementless total hip prosthesis in patients aged < 50 years at the time of their THA. METHODS: This study included 360 patients (498 hips), specifically 212 men and 148 women. The mean age of the patients at the time of their THA was 45.8 ± 8.1 years. The predominant diagnosis was osteonecrosis (56%). Demographic data, the Harris hip score, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and the University of California, Los Angeles (UCLA) activity score were recorded. Radiographic evaluation and dual-energy X-ray absorptiometry (DEXA) scanning were used to evaluate implant fixation, bone remodelling, and osteolysis. The mean follow-up was 25.2 year (range 21-28 years). RESULTS: At the latest follow-up, the mean Harris hip, WOMAC, and UCLA activity scores were 93, 10, and 6.7 points, respectively. No patients had thigh pain. All hips had osseous integration of the acetabular and femoral components. No patient had grade 3 stress shielding. The 28-year survival rate was 98.2% (95% confidence interval [CI] 95-100%) for the acetabular components and 98.8% (95% CI 95-100%) for the femoral components. Overall, 90% of the patients were satisfied with the THA results. CONCLUSION: The results suggest that a metaphyseal-engaging anatomic cementless femoral stem with alumina-on-alumina ceramic articulation provide outstanding long-term fixation and substantial pain relief well into the 3rd decade after surgery. Furthermore, there was no alumina ceramic fracture or osteolysis. Moreover, approximately 90% of the patients were satisfied with the results of their THA.

Prevalence and Risk Factors of Self-Reported Sarcopenia and Its Association With Multimorbidity in Community-Dwelling Older Adults: A Cross-Sectional Study.

This cross-sectional study aimed to identify the impact of multimorbidity on self-reported sarcopenia and compare the differences in the prevalence and associated factors of sarcopenia in community-dwelling older adults with and without multimorbidity. We enrolled 876 community-dwelling older adults in South Korea. Multimorbidity was defined as the coexistence of two or more chronic diseases, and sarcopenia was evaluated using the Strength, Assistance in walking, Rising from a chair, Climbing stairs, and Falling Questionnaire. Binomial logistic regression analyses were performed. Our study revealed that the prevalence of sarcopenia was significantly higher (43.5%) in older adults with multimorbidity than in those without multimorbidity (20.8%). This finding underscores the significant impact of multimorbidity on sarcopenia. We also found that poor perceived health status was the strongest predictor of sarcopenia in older adults with multimorbidity, while oldest-old age (≥ 85 years) was the strongest predictor of sarcopenia in those without multimorbidity. Large prospective cohort studies using objective sarcopenia screening tools are needed to demonstrate the synergistic effects of multimorbidity and sarcopenia on health outcomes among community-dwelling older adults.

Factors Affecting Adherence to Social Distancing among Adults Aged 19-44 Years: Insights from a Nationwide Survey during COVID-19 Pandemic.

Background and Objectives: Before COVID-19 vaccinations became available, adhering to non-pharmaceutical interventions (NPIs), like social distancing (SD), wearing masks, and hand hygiene, were crucial to mitigating viral spread. Many studies reported that younger individuals were more reluctant to follow these measures compared with older ones. We hypothesized that it would be worthwhile to find factors that influenced SD compliance among young people during the pre-vaccination phase of a pandemic. Materials and Methods: We analyzed data of adults aged 19-44 from the 2020 South Korean Community Health Survey and compared socio-demographic, health-related behavioral, and psychological factors between compliant and non-compliant cohorts. Results: A total of 59,943 participants were enrolled and we found that older age groups (30-39 and 40-44) and safety concerns (such as viral infection, virus-related death, economic damage, and transmitting virus to vulnerable people) were significantly associated with adherence to SD. Conversely, participants who were not living with a spouse, were unable to stay at home despite symptoms, smoked, drank, and had a negative attitude toward government policy statistically correlated with non-compliance. Conclusions: In times when NPIs were the primary defense against the pandemic, it is essential to identify factors that positively or negatively affect individual compliance with them, especially among young people. Using a large-scale, well-designed national survey, we could gain insights into the early recognition of risk factors for non-compliance and appropriate follow-up interventions (i.e., education campaigns, clear communication of public guidelines, and implementation of guidelines), which will help people to avoid suffering from other waves of future infectious diseases.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

Comparison of postoperative nausea and vomiting between Remimazolam and Propofol in Patients undergoing oral and maxillofacial surgery: a prospective Randomized Controlled Trial.

BACKGROUND: Remimazolam is a recently approved, ultra-short-acting benzodiazepine. However, few studies have investigated remimazolam in relation to postoperative nausea and vomiting (PONV). This study aimed to compare the effects of remimazolam and propofol on PONV in patients undergoing oral and maxillofacial surgery. METHODS: Patients (n = 206) aged 19-65 years who were scheduled for oral and maxillofacial surgery were randomized into two groups, the remimazolam (R) and propofol group (P). In the R group (n = 94), remimazolam was used to induce anesthesia at 12 mg/kg/h and to maintain anesthesia at 1-2 mg/kg/h. In the P group (n = 95), anesthesia was induced and maintained with propofol (target effect-site concentration: 3-5 µg/ml). In both groups, remifentanil was administered at a target effect-site concentration of 2.5-4 ng/ml. The primary outcome was the overall incidence of PONV during the first 24 h after surgery. Secondary outcomes included the severity of nausea, use of rescue antiemetics, severity of postoperative pain, use of rescue analgesia, and quality of recovery. RESULTS: The incidence of PONV during the first 24 h after surgery was 11.7% and 10.5% in the R group and P group, respectively, and there was no significant difference in the severity of nausea (P > 0.05). Ten patients in the R group and ten patients in the P group required rescue antiemetics during the first 24 h after surgery (P = 0.98). No inter-group differences were observed in terms of postoperative pain score, use of rescue analgesia, and quality of recovery (P > 0.05). CONCLUSIONS: In this study, remimazolam did not increase the incidence and severity of PONV compared with propofol. TRIAL REGISTRATION: KCT0006965, Clinical Research Information Service (CRIS), Republic of Korea. Registration date: 26/01/2022.

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

Systemic Analgesic Effects of Electroacupuncture in Healthy Individuals: Thermal Threshhold, Acupuncture Sensation Intensity and De-qi on Quantitative Sensory Test.

Background: Electroacupuncture (EA) has been known to exert analgesic effects according to several reports, but studies investigating the analgesic effect of EA using the quantitative sensory test (QST) are rare. Primary Study Objective: To investigate the analgesic effects of electroacupuncture through changes in thermal thresholds measured using the QST. Design: Pilot, randomized, single-blind, parallel design. Setting: The study was conducted at Dongguk University Bundang Oriental Hospital (DUBOH) in South Korea. Participants: We included 40 healthy participants age 20 to 40 years. Intervention: The EA group received EA for 30 minutes at 6 acupuncture points (LI4, PC6, LI10, LI11, ST36, and SP6) and the control group just rested. Outcome measures: The primary outcome measure was 4 thermal thresholds including warm detection (WDT), cold detection (CDT), hot pain (HPT), and cold pain (CPT) measured using QST at baseline and after 15, 30 and 60 minutes. The secondary outcomes were the intensity of acupuncture sensation (visual analogue scale [VAS]) and De-qi (Massachusetts General Hospital Acupuncture Sensation Scale [MASS]). Results: The EA group showed significant changes in HPT (P < .001) and CPT (P = .049) compared with the control group, whereas WDT and CDT did not significantly differ. Furthermore, the changes in thermal thresholds were more pronounced in the higher intensity acupuncture sensation group (VAS ≥40) than in the lower intensity group (VAS < 40), although not significantly. The high De-qi group presented greater changes in WDT, CDT, HPT and CPT than the low De-qi group, as measured using MASS. It was especially statistically significant at HPT a feeling of "heaviness" and "dull pain" and at CDT of "tingling." We observed no adverse events related to the study. Conclusion: The change in thermal pain thresholds effected by EA supports the analgesic effect of EA reported in previous studies. The underlying mechanisms need to be holistically considered, and further studies are needed for definitive evidence.

Minimum 30-Year Results of Bilaterally Implanted Cemented and Cementless Total Hip Arthroplasty in Patients Younger Than 50 Years.

BACKGROUND: The rate of failure of cemented and cementless total hip arthroplasty (THA) in younger patients is higher than that in elderly patients. The purpose of this study is to document the long-term clinical results of THA with the so-called third-generation cementing and the results of second-generation cementless THA in patients <50 years of age. METHODS: This study included 106 patients who had had bilateral THA with a cemented stem in one hip and a cementless stem in the other. There were 78 men and 28 women. Their mean age was 47 years (range, 21-49). The average follow-up duration was 31 years (range, 30-32.5). RESULTS: There were similar mean Harris Hip Scores (90 versus 91 points) between the groups at the final follow-up. Forty-six acetabular components (43%) in the cemented group and 48 acetabular components (45%) in the cementless group were revised. Five femoral components (5%) in the cemented group and 4 femoral components (4%) in the cementless group were revised. Survivorship of the acetabular component at 30.8 years was similar in both groups (57% in the cemented group versus 55% in the cementless group). Survivorship of the femoral component at 30.8 years was also similar in both groups (95% in the cemented group versus 96% in the cementless group). CONCLUSION: Long-term fixation of the cemented or cementless femoral stem was outstanding. There was a high rate of the acetabular component revision due to conventional polyethylene wear and periacetabular osteolysis in both hybrid and fully cementless THA groups.

Long-Term Results (Minimum of 20 Years) of a Pure Proximal-Loading Metaphyseal-Fitting Anatomic Cementless Stem Without Distal Stem Fixation in Hip Arthroplasty.

BACKGROUND: There are no reported results for more than 20 years of a pure proximal-loading anatomic cementless femoral stem without diaphyseal stem fixation. The purpose of this study was to evaluate the long-term (minimum 20 years) clinical results, bone remodeling, revision rate, and survivorship of these implants in patients aged less than 60 years. METHODS: We included 523 patients (657 hips), including 319 men and 204 women. The mean body mass index was 26.7 (range, 23-29 kg/m2). The mean age of patients at index surgery was 55 years (range, 20-59 years). The Harris Hip Score, the Western Ontario and McMaster Universities Osteoarthritis Index, and the University of California, Los Angeles activity score were recorded preoperatively and at each follow-up. Mean follow-up was 23.5 years (range, 20-27 years). RESULTS: The Harris Hip Score at the final follow-up was a mean 93 points (range, 70-100 points). The Western Ontario and McMaster Universities Osteoarthritis Index and University of California, Los Angeles activity scores at the final follow-up were 16 and 7.6 points, respectively. Five femoral components (0.8%) and 13 acetabular components (2.0%) were revised. All cases in the current series had grade 2 stress shielding; no hips had grade 3 or 4 stress shielding. Kaplan-Meier survivorship of the implants at 23.5 years was 98.0% (95% confidence interval 92%-100%) for the acetabular component and 99.2% (95% confidence interval 93%-100%) for the femoral component. CONCLUSION: A pure proximal-loading metaphyseal-fitting anatomic cementless stem with alumina-on-alumina ceramic bearing couples functioned well, with no osteolysis or mild stress-shielding at an average 23.5-year follow-up.

Is Highly Cross-Linked Polyethylene Safe for Use in High-Flexion Posterior Stabilized Total Knee Arthroplasty?

BACKGROUND: Application of highly cross-linked polyethylene (HXLPE) to a posterior cruciate-substituting total knee arthroplasty (TKA) might add the risk of fracture and failure of the tibial polyethylene insert. The purpose of this study is to evaluate the long-term (up to 19 years) clinical and radiographic results of posterior cruciate-substituting TKAs with HXLPE or conventional polyethylene. METHODS: This study analyzed the results of 1,217 patients (444 men and 773 women; mean age of 65 ± 7 years, range, 31-85) (2,434 knees) who had received a NexGen LPS-Flex prosthesis with a conventional tibial insert in one knee and the same prosthesis with an HXLPE tibial insert in the contralateral knee. The mean duration of follow-up was 17 years (range, 15-19). RESULTS: The 2 groups did not differ significantly (P > .05) with regard to the clinical and radiographic results. No knee in either group had a fracture of the tibial polyethylene post or failure of the locking mechanism of the tibial polyethylene insert or osteolysis. Twenty-eight knees (2.3%) in the HXLPE group and 26 knees (2.1%) in the conventional polyethylene group were revised. The estimated survival rate at 17 years was 97.7% in the HXLPE group and 97.9% in the conventional polyethylene group. CONCLUSION: The data suggest that clinical and radiographic findings at a mean of 17 years after posterior cruciate-substituting TKA are the same for patients treated with HXLPE and those treated with conventional polyethylene.

Annexin A2 Stabilizes Oncogenic JAG1 Intracellular Domain by Inhibiting Proteasomal Degradation in Glioblastoma Cells.

Glioblastoma (GBM) is the most lethal brain cancer, causing inevitable deaths of patients owing to frequent relapses of cancer stem cells (CSCs). The significance of the NOTCH signaling pathway in CSCs has been well recognized; however, there is no NOTCH-selective treatment applicable to patients with GBM. We recently reported that Jagged1 (JAG1), a NOTCH ligand, drives a NOTCH receptor-independent signaling pathway via JAG1 intracellular domain (JICD1) as a crucial signal that renders CSC properties. Therefore, mechanisms regulating the JICD1 signaling pathway should be elucidated to further develop a selective therapeutic regimen. Here, we identified annexin A2 (ANXA2) as an essential modulator to stabilize intrinsically disordered JICD1. The binding of ANXA2 to JICD1 prevents the proteasomal degradation of JICD1 by heat shock protein-70/90 and carboxy-terminus of Hsc70 interacting protein E3 ligase. Furthermore, JICD1-driven propagation and tumor aggressiveness were inhibited by ANXA2 knockdown. Taken together, our findings show that ANXA2 maintains the function of the NOTCH receptor-independent JICD1 signaling pathway by stabilizing JICD1, and the targeted suppression of JICD1-driven CSC properties can be achieved by blocking its interaction with ANXA2.

Exploring the Knowledge Structure of Patient Safety in Nursing Using a Keyword Network Analysis.

Patient safety is a critical and long-standing issue in nursing research. The purposes of this study were to explore the knowledge structure of patient safety and to provide a direction for future research by offering new perspectives and a theoretical clarification of patient safety in nursing. Keyword network analysis was performed by extracting keywords from abstracts of 6072 published articles. To reflect nursing perspectives, focus group interviews were conducted and Kim's typology consisting of four domains was used as the framework of analysis. Visualized knowledge structure showed avoiding medication error and preventing pressure ulcers or falls remain important topics within this research field. The distribution of core keywords as per four domains was in the following order: practice, client, environment, and client-nurse domain. Within the client domain, patients' harm-related core keywords were limited to physical harm. The detailed knowledge structure consisted of five themes: patient, preventable patient harm, practice, error, and environment. It comprised risk assessment for patients' characteristics and environmental elements surrounding patient and nursing practice, and risk management using information as knowledge-based nursing practice. Regarding further research, we suggest a multidimensional approach to patient harm, and the utilization of the client-nurse relationship and information systems as strategies for patient safety.

A rapid ELISA for the detection of matrix metallopeptidase 9 using a recombinant Fab-type antibody.

Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases.

Development of a CHO cell line for stable production of recombinant antibodies against human MMP9.

BACKGROUND: Human matrix metalloproteinase 9 (hMMP9) is a biomarker in several diseases, including cancer, and the need for developing detectors and inhibitors of hMMP9 is increasing. As an antibody against hMMP9 can be selectively bound to hMMP9, the use of anti-MMP9 antibody presents new possibilities to address hMMP9-related diseases. In this study, we aimed to establish a stable Chinese hamster ovary (CHO) cell line for the stable production of antibodies against hMMP9. RESULTS: Weconstructed recombinant anti-hMMP9 antibody fragment-expressing genes and transfected these to CHO cells. We chose a single clone, and successfully produced a full-sized antibody against hMMP9 with high purity, sensitivity, and reproducibility. Subsequently, we confirmed the antigen-binding efficiency of the antibody. CONCLUSIONS: We developed a novel recombinant anti-hMMP9 antibody via a CHO cell-based mammalian expression system, which has a high potential to be used in a broad range of medical and industrial areas.

Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.

Skin atrophy caused by topical glucocorticoids is less common in patients with atopic dermatitis than in those with psoriasis.

Although the long-term use of topical glucocorticoids (TGC) may induce skin atrophy including striae distensae (SD), patients with atopic dermatitis (AD) appear to have lesser degree of skin atrophy than those with psoriasis (PSO). Periostin, encoded by POSTN, is involved in tissue remodelling processes of chronic AD lesions. This study was designed to investigate the difference in the occurrence of skin atrophy in patients with AD or PSO when treated with TGC and to elucidate the association between skin atrophy and periostin. Big data analysis using Korean Health Claims Database was performed to determine the prevalence of SD in AD and PSO patients. Blood and skin eosinophils count and dermal fibrosis between AD and PSO patients were compared, and immunohistochemistry for periostin and mRNA sequencing in the dermis were performed. Animal experiments using AD and PSO murine model were conducted. Big data analysis revealed that patients with AD have significantly lesser degree of SD than patients with PSO. The ratio of the dermal fibrous tissues and eosinophil counts were significantly higher in AD patients. In AD skin, periostin was more widely distributed in the entire dermis and POSTN mRNAs were significantly upregulated. Dermal thickness and fibrosis were significantly higher in AD mice even after TGC treatment. A significant positive correlation was observed between dermal fibrosis and tissue eosinophil counts. Lesser skin atrophy in AD patients even after long-term TGC application could be resulted from skin fibrosis caused by increased tissue eosinophils and periostin deposition.

Constructing multi-enzymatic cascade reactions for selective production of 6-bromoindirubin from tryptophan in Escherichia coli.

6-Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin-derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site-specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6-halogenase fused with flavin reductase Fre (Fre-L3-SttH), tryptophanase (TnaA), toluene 4-monooxygenase (PmT4MO), and flavin-containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6-bromoindole with 45% yield to produce 6-bromo-2-oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen.

Enhancing biosynthesis of 2'-Fucosyllactose in Escherichia coli through engineering lactose operon for lactose transport and α -1,2-Fucosyltransferase for solubility.

2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and one of the most actively studied human milk oligosaccharides (HMOs). When 2'-FL is produced through biological production using a microorganism, like Escherichia coli, d-lactose is often externally fed as an acceptor substrate for fucosyltransferase (FT). When d-glucose is used as a carbon source for the cell growth and d-lactose is transported by lactose permease (LacY) in lac operon, d-lactose transport is under the control of catabolite repression (CR), limiting the supply of d-lactose for FT reaction in the cell, hence decreasing the production of 2'-FL. In this study, a remarkable increase of 2'-FL production was achieved by relieving the CR from the lac operon of the host E. coli BL21 and introducing adequate site-specific mutations into α-1,2-FT (FutC) for enhancement of catalytic activity and solubility. For the host engineering, the native lac promoter (Plac ) was substituted for tac promoter (Ptac ), so that the lac operon could be turned on, but not subjected to CR by high d-glucose concentration. Next, for protein engineering of FutC, family multiple sequence analysis for conserved amino acid sequences and protein-ligand substrate docking analysis led us to find several mutation sites, which could increase the solubility of FutC and its activity. As a result, a combination of four mutation sites (F40S/Q150H/C151R/Q239S) was identified as the best candidate, and the quadruple mutant of FutC enhanced 2'-FL titer by 2.4-fold. When the above-mentioned E. coli mutant host transformed with the quadruple mutant of futC was subjected to fed-batch culture, 40 g l-1 of 2'-FL titer was achieved with the productivity of 0.55 g l-1 h-1 and the specific 2'-FL yield of 1.0 g g-1 dry cell weight.