Dissecting polyunsaturated fatty acid synthases for product profile control

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans and are ingested from fish oils. Because of increasing demand, however, fermentative processes using microalgae, yeasts, and fungi have been developed to produce DHA, EPA, and ARA, respectively. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts even though the multiple catalytic domains in each huge subunit are very similar. In this study, we carefully dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles (Figure 1) 1). Moreover, for the first time, we converted a practical microalgal DHA synthase into an EPA synthase based on the obtained results 2). Please click Additional Files below to see the full abstract

Cloning, Sequencing, and Functional Analysis of the Biosynthetic Gene Cluster of Macrolactam Antibiotic Vicenistatin in Streptomyces halstedii

AbstractVicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning ∼64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well

Enhanced Recombinant Protein Productivity by Genome Reduction in Bacillus subtilis

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production

Prediction of stroke patients’ bedroom-stay duration: machine-learning approach using wearable sensor data

Background: The importance of being physically active and avoiding staying in bed has been recognized in stroke rehabilitation. However, studies have pointed out that stroke patients admitted to rehabilitation units often spend most of their day immobile and inactive, with limited opportunities for activity outside their bedrooms. To address this issue, it is necessary to record the duration of stroke patients staying in their bedrooms, but it is impractical for medical providers to do this manually during their daily work of providing care. Although an automated approach using wearable devices and access points is more practical, implementing these access points into medical facilities is costly. However, when combined with machine learning, predicting the duration of stroke patients staying in their bedrooms is possible with reduced cost. We assessed using machine learning to estimate bedroom-stay duration using activity data recorded with wearable devices.Method: We recruited 99 stroke hemiparesis inpatients and conducted 343 measurements. Data on electrocardiograms and chest acceleration were measured using a wearable device, and the location name of the access point that detected the signal of the device was recorded. We first investigated the correlation between bedroom-stay duration measured from the access point as the objective variable and activity data measured with a wearable device and demographic information as explanatory variables. To evaluate the duration predictability, we then compared machine-learning models commonly used in medical studies.Results: We conducted 228 measurements that surpassed a 90% data-acquisition rate using Bluetooth Low Energy. Among the explanatory variables, the period spent reclining and sitting/standing were correlated with bedroom-stay duration (Spearman’s rank correlation coefficient (R) of 0.56 and −0.52, p < 0.001). Interestingly, the sum of the motor and cognitive categories of the functional independence measure, clinical indicators of the abilities of stroke patients, lacked correlation. The correlation between the actual bedroom-stay duration and predicted one using machine-learning models resulted in an R of 0.72 and p < 0.001, suggesting the possibility of predicting bedroom-stay duration from activity data and demographics.Conclusion: Wearable devices, coupled with machine learning, can predict the duration of patients staying in their bedrooms. Once trained, the machine-learning model can predict without continuously tracking the actual location, enabling more cost-effective and privacy-centric future measurements

A Small Bowel Ulcer due to Clopidogrel with Cytomegalovirus Enteritis Diagnosed by Capsule and Double-Balloon Endoscopy

We report the first case of small bowel ulcers due to clopidogrel in a 74-year-old man. He presented with diarrhea and melena after having been taking low-dose aspirin (LDA) and clopidogrel. There was no evidence of bleeding in the stomach, duodenum, or colon. Capsule endoscopy showed multiple ulcers and erosions in the small intestine. Double-balloon endoscopy revealed multiple ulcers throughout the ileum. Examination of the biopsy specimen showed cytomegalovirus infection. His LDA was discontinued and he was prescribed ganciclovir. However, the small bowel ulcers were aggravated. Therefore, clopidogrel was discontinued. The small bowel ulcers subsequently healed completely, forming scars

Thread-Traction with a Sheath of Polypectomy Snare Facilitates Endoscopic Submucosal Dissection of Early Gastric Cancers

Although the thread-traction (TT) method has been found useful during endoscopic submucosal dissection (ESD) for early gastric cancers, the movement of the thread interferes with the movement of the endoscope, and the lesion can only be pulled to the mouth side. We have developed the novel TT method using a sheath of polypectomy snare (TTSPS). The TTSPS method enables free and independent movement of the thread and the endoscope and allows pulling the lesion towards the anal as well as oral side. The median dissection times, numbers of instances of arterial bleeding, and numbers of local injections into the submucosal layer were significantly lower for ESD with TTSPS than for conventional ESD. Countertraction ESD using the TTSPS method is straightforward, safe, easy, noninvasive, and cost effective, and it uses instruments readily available in most hospitals to enhance visualization of cutting lines. Therefore, the TTSPS method can be universally applied in conventional ESD

Efficacy of Neuraminidase Inhibitors against H5N6 Highly Pathogenic Avian Influenza Virus in a Nonhuman Primate Model.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used nonhuman primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe, with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in interferon alpha (IFN-α) showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and the severity of symptoms in the early stage. This study also showed pathologically severe lung injury states in cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and further studies on virus pathogenicity and new antiviral therapies

MHC matching improves engraftment of iPSC-derived neurons in non-human primates.

The banking of human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cells (iPSCs) is considered a future clinical strategy for HLA-matched cell transplantation to reduce immunological graft rejection. Here we show the efficacy of major histocompatibility complex (MHC)-matched allogeneic neural cell grafting in the brain, which is considered a less immune-responsive tissue, using iPSCs derived from an MHC homozygous cynomolgus macaque. Positron emission tomography imaging reveals neuroinflammation associated with an immune response against MHC-mismatched grafted cells. Immunohistological analyses reveal that MHC-matching reduces the immune response by suppressing the accumulation of microglia (Iba-1+) and lymphocytes (CD45+) into the grafts. Consequently, MHC-matching increases the survival of grafted dopamine neurons (tyrosine hydroxylase: TH+). The effect of an immunosuppressant, Tacrolimus, is also confirmed in the same experimental setting. Our results demonstrate the rationale for MHC-matching in neural cell grafting to the brain and its feasibility in a clinical setting.Major histocompatibility complex (MHC) matching improves graft survival rates after organ transplantation. Here the authors show that in macaques, MHC-matched iPSC-derived neurons provide better engraftment in the brain, with a lower immune response and higher survival of the transplanted neurons

Characterization of tumour-infiltrating lymphocytes in a tumour rejection cynomolgus macaque model.

Immunotherapy has emerged as a promising and effective treatment for cancer, yet the clinical benefit is still variable, in part due to insufficient accumulation of immune effector cells in the tumour microenvironment. Better understanding of tumour-infiltrating lymphocytes (TILs) from nonhuman primate tumours could provide insights into improving effector cell accumulation in tumour tissues during immunotherapy. Here, we characterize TILs in a cynomolgus macaque tumour model in which the tumours were infiltrated with CD4+ and CD8+ T cells and were eventually rejected. The majority of CD4+ and CD8+ TILs exhibited a CD45RA-CCR7- effector memory phenotype, but unlike circulating T cells, they expressed CD69, a marker for tissue-resident memory T (TRM) cells. CD69-expressing CD8+ TILs expressed high levels of the cytotoxic molecule granzyme B and the co-inhibitory receptor PD-1. Consistent with the TRM cell phenotype, CD8+ TILs minimally expressed CX3CR1 but expressed CXCR3 at higher levels than circulating CD8+ T cells. Meanwhile, CXCL9, CXCL10 and CXCL11, chemokine ligands for CXCR3, were expressed at high levels in the tumours, thus attracting CXCR3+CD8+ T cells. These results indicate that tumour-transplanted macaques can be a useful preclinical model for studying and optimizing T cell accumulation in tumours for the development of new immunotherapies