Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (kcat/Km) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25 °C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2 g/l cells, and 7 mM ARA as substrate in a 500 ml-Erlenmeyer baffled flask with 50 ml reaction solution with agitation at 200 rpm in the presence of 10 mM cysteine as a reduction agent, respectively. Under these conditions, 6.4 mM 9S-hydroxyeicosatetraenoic acid, 6.2 mM 9S-hydroxyeicosapentaenoic acid, and 5.9 mM 11S-hydroxydocosahexaenoic acid were produced in 30 min, 40 min, and 60 min with specific productivities of 1067 µmol/min/g, 775 µmol/min/g, and 492 µmol/min/g, volumetric productivities of 213 µM/min, 155 µM/min, and 98 µM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.

Biocatalytic synthesis of dihydroxy fatty acids as lipid mediators from polyunsaturated fatty acids by double dioxygenation of the microbial 12S-lipoxygenase.

Leukotrienes (LTs) and maresins (MaRs) are human lipid mediators (LMs) involved in immune response and anti-inflammation, respectively. These compounds and their isomers are generated in trace amounts by lipoxygenases (LOXs) in human macrophages and neutrophils. These LMs have been synthesized using nonenvironmentally benign synthetic protocols, which are expensive. 8S- and 15S-LOXs with double dioxygenating activities have previously been reported, whereas 12S-LOX with double dioxygenating activity have not been reported to date. Here, we discovered a wild-type 12S-LOX with double dioxygenating activity from the bacterium Endozoicomonas numazuensis, which produced dihydroxy fatty acids (DiHFAs) as LMs from polyunsaturated fatty acids via double dioxygenation. The enzyme activity for producing DiHFA was approximately 550-fold higher than that of mammalian LOX with double dioxygenating activity. The microbial 12S-LOX converted 3.00 mM of arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid to 2.37 mM (797 mg/L) 6-trans-8-cis-12S-epimer of LTB4, 1.59 mM (532 mg/L) 6-trans-8-cis-12S-epimer of LTB5, 1.35 mM (498 mg/L) 10-cis-12-trans-7S-epimer of MaR1n-3 DPA , and 1.54 mM (555 mg/L) 10-cis-12-trans-7S-epimer of MaR1 within 2 h, which were 5.3-, 7.6-, 3.1-, and 5.5-fold higher than those biosynthesized by the previously reported microbial engineered 12S-LOX with double dioxygenating activity, respectively. These findings contribute to the efficient and environmentally friendly biosynthesis of LMs and stimulate physiological study on LMs.

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα-PGC-1α-ERRα Circuit after Traumatic Brain Injury.

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)-estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O2 consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα-PGC-1α-ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα-PGC-1α-ERRα circuit and consequent oxidative phosphorylation, O2 consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.

Case report: Abdominal hernia repair using a surgical wire and an autologous omental graft in a Japanese Black calf.

Background: Hernia formation is a well-known abdominal wall disorder in calves and most often occurs in the umbilical region. In addition, it occurs in the abdominal wall outside the umbilical region. It has been reported to involve acquired factors, such as external force to the lower or lateral abdominal wall, trauma, muscle weakness, and, although rare, congenital or hereditary factors. Although there have been reports on the repair of abdominal wall hernias caused by abnormal muscle formation, there have been no reports on the treatment of abdominal wall hernias caused by abnormal vertebral and rib formation in cattle or other ruminants. In this study, for the first time, we describe a case of successful closure of a hernia in the left flank caused by malformation of the vertebrae and ribs. The repair was performed by narrowing the ribs using a surgical wire and covering the defect with a free omental graft. Case presentation: A male Japanese Black calf showed a mild bulge of the left abdominal wall and abnormal morphology of the left ribs immediately after birth. At 3 months of age, computed tomography revealed fusion of the 9th, 10th, and 11th thoracic vertebrae and missing formation of the 10th and 11th left ribs at the thoracic vertebral fusion site. Additionally, a 15.2 × 24.4 cm abdominal hernia had formed in the abdominal wall. During surgery, the ribs forming the hernia were narrowed with a surgical wire, and the hernia was covered with a large autologous free omental graft to prevent intra-abdominal organ prolapse. At the monthly follow-up (11 months after surgery), the calf had developed similarly to other calves of the same age on the farm, and no complications were noted. Conclusions: The current case shows that a combination of rib correction using a surgical wire combined with transplantation of a free autologous greater omentum graft was shown to be a possible treatment option for surgical repair of large abdominal wall hernias caused by rib malformation.

Repair of a calcaneal fracture with a combination of internal and external fixation in a calf.

A 6-day-old male Japanese Black calf presented with a transverse fracture of the left calcaneus. In calcaneal fractures, traction of the gastrocnemius muscle causes substantial displacement of the proximal fracture fragment; therefore, external fixation alone is prone to failure of fusion or deformed fusion. Furthermore, internal fixation alone may result in refracture due to the high load on the implant. Therefore, internal and external fixation were used to treat the fracture. Bone fusion was observed on postoperative day 50; the wire was removed on postoperative day 90. Radiographic examination at 4 months postoperatively revealed that the bone had fused in normal alignment. Therefore, a good prognosis can be expected for calcaneal fractures treated with combined internal and external fixation.

A retrospective analysis for criteria of surgical intervention in Japanese Black calves with respiratory signs caused by perinatal rib fracture.

Medical records of 16 Japanese Black calves with respiratory signs associated with perinatal rib fracture were analyzed, retrospectively, to speculate criteria of surgical intervention for the disease. For this analysis, the severity of respiratory condition was classified into three grades: Grade 1, with no wheezing, Grade 2, with wheezing after excitement but not at rest and Grade 3, with wheezing at rest. Grade 1 (n=3) received only conservative management. Seven of Grade 2 (n=8) and all of Grade 3 (n=5) had surgical management. Clinical outcome was good in all of Grade 1, and the surgical success rate was 83% in other groups. Our result suggests that surgical intervention should be considered for cases with more the severity of Grade 2.

CTCF controls three-dimensional enhancer network underlying the inflammatory response of bone marrow-derived dendritic cells.

Dendritic cells are antigen-presenting cells orchestrating innate and adaptive immunity. The crucial role of transcription factors and histone modifications in the transcriptional regulation of dendritic cells has been extensively studied. However, it is not been well understood whether and how three-dimensional chromatin folding controls gene expression in dendritic cells. Here we demonstrate that activation of bone marrow-derived dendritic cells induces extensive reprogramming of chromatin looping as well as enhancer activity, both of which are implicated in the dynamic changes in gene expression. Interestingly, depletion of CTCF attenuates GM-CSF-mediated JAK2/STAT5 signaling, resulting in defective NF-κB activation. Moreover, CTCF is necessary for establishing NF-κB-dependent chromatin interactions and maximal expression of pro-inflammatory cytokines, which prime Th1 and Th17 cell differentiation. Collectively, our study provides mechanistic insights into how three-dimensional enhancer networks control gene expression during bone marrow-derived dendritic cells activation, and offers an integrative view of the complex activities of CTCF in the inflammatory response of bone marrow-derived dendritic cells.

Prophylactic role of Korean Red Ginseng in astrocytic mitochondrial biogenesis through HIF-1α.

Background: Korean Red Ginseng extract (KRGE) has been used as a health supplement and herbal medicine. Astrocytes are one of the key cells in the central nervous system (CNS) and have bioenergetic potential as they stimulate mitochondrial biogenesis. They play a critical role in connecting the brain vasculature and nerves in the CNS. Methods: Brain samples from KRGE-administered mice were tested using immunohistochemistry. Treatment of human brain astrocytes with KRGE was subjected to assays such as proliferation, cytotoxicity, Mitotracker, ATP production, and O2 consumption rate as well as western blotting to demonstrate the expression of proteins related to mitochondria functions. The expression of hypoxia-inducible factor-1α (HIF-1α) was diminished utilizing siRNA transfection. Results: Brain samples from KRGE-administered mice harbored an increased number of GFAP-expressing astrocytes. KRGE triggered the proliferation of astrocytes in vitro. Enhanced mitochondrial biogenesis induced by KRGE was detected using Mitotracker staining, ATP production, and O2 consumption rate assays. The expression of proteins related to mitochondrial electron transport was increased in KRGE-treated astrocytes. These effects were blocked by HIF-1α knockdown. The factors secreted from KRGE-treated astrocytes were determined, revealing the expression of various cytokines and growth factors, especially those related to angiogenesis and neurogenesis. KRGE-treated astrocyte conditioned media enhanced the differentiation of adult neural stem cells into mature neurons, increasing the migration of endothelial cells, and these effects were reduced in the background of HIF-1α knockdown. Conclusion: Our findings suggest that KRGE exhibits prophylactic potential by stimulating astrocyte mitochondrial biogenesis through HIF-1α, resulting in improved neurovascular function.

Dual Effects of Korean Red Ginseng on Astrocytes and Neural Stem Cells in Traumatic Brain Injury: The HO-1-Tom20 Axis as a Putative Target for Mitochondrial Function.

Astrocytes display regenerative potential in pathophysiologic conditions. In our previous study, heme oxygenase-1 (HO-1) promoted astrocytic mitochondrial functions in mice via the peroxisome-proliferator-activating receptor-γ coactivator-1α (PGC-1α) pathway on administering Korean red ginseng extract (KRGE) after traumatic brain injury (TBI). In this study, KRGE promoted astrocytic mitochondrial functions, assessed with oxygen consumption and adenosine triphosphate (ATP) production, which could be regulated by the translocase of the outer membrane of mitochondria 20 (Tom20) pathway with a PGC-1α-independent pathway. The HO-1-Tom20 axis induced an increase in mitochondrial functions, detected with cytochrome c oxidase subunit 2 and cytochrome c. HO-1 crosstalk with nicotinamide phosphoribosyltransferase was concomitant with the upregulated nicotinamide adenine dinucleotide (NAD)/NADH ratio, thereby upregulating NAD-dependent class I sirtuins. In adult neural stem cells (NSCs), KRGE-treated, astrocyte-conditioned media increased oxygen consumption and Tom20 levels through astrocyte-derived HO-1. HO inactivation by Sn(IV) protoporphyrin IX dichloride in TBI mice administered KRGE decreased neuronal markers, together with Tom20. Thus, astrocytic HO-1 induced astrocytic mitochondrial functions. HO-1-related, astrocyte-derived factors may also induce neuronal differentiation and mitochondrial functions of adult NSCs after TBI. KRGE-mediated astrocytic HO-1 induction may have a key role in repairing neurovascular function post-TBI in peri-injured regions by boosting astrocytic and NSC mitochondrial functions.

Breathing Pattern Analysis in Cattle Using Infrared Thermography and Computer Vision.

Breathing patterns can be considered a vital sign providing health information. Infrared thermography is used to evaluate breathing patterns because it is non-invasive. Our study used not only sequence temperature data but also RGB images to gain breathing patterns in cattle. Mask R-CNN was used to detect the ROI (region of interest, nose) in the cattle RGB images. Mask segmentation from the ROI detection was applied to the corresponding temperature data. Finally, to visualize the breathing pattern, we calculated the temperature values in the ROI by averaging all temperature values in the ROI. The results in this study show 76% accuracy with Mask R-CNN in detecting cattle noses. With respect to the temperature calculation methods, the averaging method showed the most appropriate breathing pattern compared to other methods (maximum temperature in the ROI and integrating all temperature values in the ROI). Finally, we compared the breathing pattern from the averaging method and that from the thermal image observation and found them to be highly correlated (R2 = 0.91). This method is not labor-intensive, can handle big data, and is accurate. In addition, we expect that the characteristics of the method might enable the analysis of temperature data from various angles.

Radiographic diagnosis and surgical management for successful outcomes for osteochondrosis in Japanese Black calves.

Osteochondrosis (OC) is not well recognized in cattle. Japanese Black (JB) cattle are valued for their beef and studies regarding OC are scarce. Hence, the aim of this study was to retrospectively discuss clinical and radiographical findings, diagnosis and treatment options for OC in JB cattle for successful outcomes. Medical records of 11 calves affected with OC were reviewed, retrospectively. Clinical and radiographic findings, treatment and outcomes and postmortem examination findings were analyzed. All calves had lameness. Stifle (4/14) and tarsal (4/14) were the most affected joints. Subchondral bone lucencies were observed in all joints (14/14) and accompanied with sclerosis (10/14). Radiographic grading was performed in calves. Calves were treated surgically (8/11) or conservatively (1/11). Surgically treated calves were auctioned (5/8). Postmortem examination was performed (2/11). Gross findings revealed ulcerative lesion at articular surface. Histopathologically, cartilage layer was defected at lesion. Inflammation (2/2) and granulation tissue (1/2) was also observed. In conclusion, OC in JB cattle can be diagnosed on the basis of radiography with history and clinical signs in clinical setting. Surgery should be performed in mild cases for successful outcomes. A modified radiographic grading criteria for clinical cases may hold potential in evaluating prognosis and outcomes.

CCCTC-binding factor is essential to the maintenance and quiescence of hematopoietic stem cells in mice.

Hematopoiesis involves a series of lineage differentiation programs initiated in hematopoietic stem cells (HSCs) found in bone marrow (BM). To ensure lifelong hematopoiesis, various molecular mechanisms are needed to maintain the HSC pool. CCCTC-binding factor (CTCF) is a DNA-binding, zinc-finger protein that regulates the expression of its target gene by organizing higher order chromatin structures. Currently, the role of CTCF in controlling HSC homeostasis is unknown. Using a tamoxifen-inducible CTCF conditional knockout mouse system, we aimed to determine whether CTCF regulates the homeostatic maintenance of HSCs. In adult mice, acute systemic CTCF ablation led to severe BM failure and the rapid shrinkage of multiple c-Kithi progenitor populations, including Sca-1+ HSCs. Similarly, hematopoietic system-confined CTCF depletion caused an acute loss of HSCs and highly increased mortality. Mixed BM chimeras reconstituted with supporting BM demonstrated that CTCF deficiency-mediated HSC depletion has both cell-extrinsic and cell-intrinsic effects. Although c-Kithi myeloid progenitor cell populations were severely reduced after ablating Ctcf, c-Kitint common lymphoid progenitors and their progenies were less affected by the lack of CTCF. Whole-transcriptome microarray and cell cycle analyses indicated that CTCF deficiency results in the enhanced expression of the cell cycle-promoting program, and that CTCF-depleted HSCs express higher levels of reactive oxygen species (ROS). Importantly, in vivo treatment with an antioxidant partially rescued c-Kithi cell populations and their quiescence. Altogether, our results suggest that CTCF is indispensable for maintaining adult HSC pools, likely by regulating ROS-dependent HSC quiescence.

Action Mechanism of Chamaecyparis obtusa Oil on Hair Growth.

This study was carried out to examine the action mechanism of Chamaecyparis obtusa oil (CO) on hair growth in C57BL/6 mice. For alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT) activities in the skin tissue, at week 4, the 3% minoxidil (MXD) and 3% CO treatment groups showed an ALP activity that was significantly higher by 85% (p < 0.001) and 48% (p < 0.05) and an γ-GT activity that was significantly higher by 294% (p < 0.01) and 254% (p < 0.05) respectively, as compared to the saline (SA) treatment group. For insulin-like growth factor-1 (IGF-1) mRNA expression in the skin tissue, at week 4, the MXD and CO groups showed a significantly higher expression by 204% (p < 0.05) and 426% (p < 0.01) respectively, as compared to the SA group. At week 4, vascular endothelial growth factor (VEGF) expression in the MXD and CO groups showed a significantly higher expression by 74% and 96% (p < 0.05) respectively, however, epidermal growth factor (EGF) expression in the MXD and CO groups showed a significantly lower expression by 66% and 61% (p < 0.05) respectively, as compared to the SA group. Stem cell factor (SCF) expression in the MXD and CO groups was observed by immunohistochemistry as significant in a part of the bulge around the hair follicle and in a part of the basal layer of the epidermis. Taking all the results together, on the basis of effects on ALP and γ-GT activity, and the expression of IGF-1, VEGF and SCF, which are related to the promotion of hair growth, it can be concluded that CO induced a proliferation and division of hair follicle cells and maintained the anagen phase. Because EGF expression was decreased significantly, CO could delay the transition to the catagen phase.