Search

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific IgG antibody lateral flow assays that enables rapid and accurate evaluation of test strips. By capturing an image with a smartphone camera, SCAISY quantitatively analyzes antibody levels and provides results to the user. We analyzed changes in antibody levels over time in over 248 individuals, including vaccine type, number of doses, and infection status, with a standard deviation of less than 10%. We also tracked antibody levels in six participants before and after SARS-CoV-2 infection. Finally, we examined the effects of lighting conditions, camera angle, and smartphone type to ensure consistency and reproducibility. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

Introduction The complications of the conventional medialized design for reverse total shoulder arthroplasty (RSA) are increased scapular notching, and decreased external rotation and deltoid wrapping. Currently, lateralization design RSA, which avoid scapular notching and improve impingement-free range of motion, is commonly used. Especially, humeral lateralization design was most commonly used and glenoid lateralization design was preferred for glenoid abnormities. We compared mid-term clinical and radiologic outcomes of glenoid and humeral lateralization RSA in an Asian population in this study. Materials and Methods We enrolled 124 shoulders of 122 consecutive patients (mean age 73.8 ± 6.8 years) who received glenoid or humeral lateralization RSA from May, 2012 to March, 2019. We divided these patients into two groups according to RSA using either glenoid or humeral lateralization design. These different designs were introduced consecutively in Korea. The clinical and radiological results of 60 glenoid lateralization RSA (Group I, 60 patients) and 64 humeral lateralization RSA (Group II, 62 patients) were retrospectively evaluated and also were compared between the two groups. All patients were followed for mean 3 years. Results The clinical and radiologic outcomes of the two groups did not differ significantly, including scapular notching (p = 0.134). However, humeral lateralization RSA showed a larger glenoid-tuberosity (GT) distance (p = 0.000) and less distalization shoulder angle (DSA) (p = 0.035). The complication rate did not differ significantly either. But, revision surgery was performed for 2 humeral loosening in the Group II. Conclusion The clinical and radiologic outcomes of the two groups did not differ significantly, including scapular notching at mid-term follow-up. However, humeral lateralization design showed larger GT distance and less DSA. Humeral lateralization design RSA could preserve the normal shoulder contour due to a larger GT distance (more lateralization) and provide less deltoid tension due to less DSA (less distalization of COR).

Background and Objectives: The majority of research on the effects of osteoporosis drugs has measured the bone mineral density (BMD) of the spine and femur through dual-energy X-ray absorptiometry (DEXA) and compared and analyzed the effects of the drugs through changes in the BMD values. This study aims to compare osteoclast and sclerostin expression in osteocytes after risedronate therapy by obtaining femoral heads from patients with hip fractures. Materials and Methods: We obtained the femoral heads of 10 female patients (age: ≥65 years) who received risedronate therapy for at least 1 year through hip arthroplasty during 2019–2021 (risedronate group). Meanwhile, 10 patients who had never received osteoporosis treatment were selected as controls using propensity scores with age, body mass index, and bone density as covariates (control group). While the osteoclast count was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, the sclerostin expression in osteocytes was assessed using immunohistochemistry. Moreover, Western blotting and polymerase chain reaction (PCR) were performed for receptor activation of nuclear factor kappa-Β ligand (RANKL), RANK, osteoprotegerin (OPG), sclerostin, and bone morphogenetic protein-2 (BMP2). Results: TRAP staining revealed significantly more TRAP-positive cells in the control group (131.75 ± 27.16/mm2) than in the risedronate group (28.00 ± 8.12/mm2). Moreover, sclerostin-positive osteocytes were expressed more in the control group (364.12 ± 28.12/mm2) than in the risedronate group (106.93 ± 12.85/mm2). Western blotting revealed that the expressions of RANKL, RANK, sclerostin, and BMP2 were higher in the control group than in the risedronate group (p < 0.05). Furthermore, RANK, sclerostin, and OPG protein levels were higher in the control group than in the risedronate group. Conclusions: In this study, the risedronate group demonstrated lower osteoclast activity and sclerostin expression in osteocytes in the femoral head than the control group.

Recurrent spillovers of α- and β-coronaviruses (CoV) such as acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, SARS-CoV-2, and possibly human CoV (NL63, 229E, OC43, and HKU1) have caused serious morbidity and mortality worldwide. Six receptor binding domains (RBDs) derived from α- and β-CoV that are considered to have originated from animals and cross-infected humans were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric subunits, PCNA1, PCNA2, and PCNA3. These were used to form a scaffold-based mosaic multivalent antigen, 6RBD-np. Electron microscopic and atomic force microscopic images show a ring-shaped disk with six protruding RBDs, like jewels in a crown, with a size of 40 nm. Prime-boost immunizations with 6RBD-np in BALB/c mice elicited strong, dose-dependent antibody responses. In human angiotensin converting enzyme 2-transgenic mice, the same immunization induced full-protection against SARS-CoV-2 wild type and Delta challenges, resulting in a 100% survival rate. The mosaic 6RBD-np provides a potential platform for developing a pan-CoV vaccine against newly emerging SARS-CoV-2 variants and future CoV spillovers.SignificanceDespite the arsenal of COVID-19 vaccines, hospitalization and mortality associated with SARS-CoV-2 (acute respiratory syndrome coronavirus 2) variants remain high. There is an urgent need to develop next-generation COVID vaccines that provide broad protection against diseases by current and newly emerging SARS-CoV-2 variants. In this study, six receptor binding domains (RBDs) derived from α- and β-CoV were linked to proliferating cell nuclear antigen (PCNA) heterotrimeric scaffolds. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens. The prime-boost immunization in BALB/c and human angiotensin converting enzyme 2-transgenic mice with the 6RBD-np elicited strong, dose-dependent antibody responses and induced full-protection against both the SARS-CoV-2 wild type (WT) and Delta challenges. This study provides proof-of-concept that the mosaic 6RBD-np induces 100% protection against SARS-CoV-2 WT and Delta. It provides the potential of co-displaying heterologous antigens for novel vaccine designs, which can be deployed countering future pandemics.

Cancer is caused by uncontrolled cell division and is a leading cause of mortality worldwide. Oenothera odorata (O. odorata) extract is used in herbal medicine to inhibit inflammation, but its potential anti-tumor properties have not been fully evaluated. Here, we demonstrated that O. odorata extract inhibits the proliferation of lung adenocarcinoma and melanoma cell lines In Vitro, and also inhibits the growth of melanoma cells In Vivo. After partitioning the extract with n-hexane, chloroform, ethyl acetate, and n-butanol, it was found that the butanol-soluble (OOB) and water-soluble (OOW) fractions of O. odorata extract are effective at inhibiting tumor cell growth In Vivo although OOW is more effective than OOB. Interestingly, these fractions did not inhibit the growth of non-cancerous cells. The anti-proliferative effects of the OOW fraction were found to be mediated by inhibition of glycolysis and cellular respiration. UPLC of both fractions showed two major common peaks, which were predicted to be hydrolyzable tannin-related compounds. Taken together, these data suggest that O. odorata extract has anti-tumor properties, and the molecular mechanism involves metabolic alterations and inhibition of cell proliferation. O. odorata extract therefore holds promise as a novel natural product for the treatment of cancer.

Sophora flavescens is used as a traditional herbal medicine to modulate inflammatory responses. However, little is known about the impact of (‐)‐maackiain, a compound derived from S. flavescens, on the activation of inflammasome/caspase‐1, a key factor in interleukin‐1β (IL‐1β) processing. Here, we report that (‐)‐maackiain potently amplified caspase‐1 cleavage in macrophages in response to nigericin (Nig). In macrophages primed with either lipopolysaccharide or monophosphoryl lipid A, Nig‐mediated caspase‐1 cleavage was also markedly promoted by (‐)‐maackiain. Notably, (‐)‐maackiain induced the production of vimentin, an essential mediator for the activation of the NOD‐, LRR‐, and pyrin domain‐containing protein 3 inflammasome, thereby contributing to promotion of the formation of the inflammasome complex to activate caspase‐1. Taken together, our data suggest that (‐)‐maackiain exerts an immunostimulatory effect by promoting IL‐1β production via activation of the inflammasome/caspase‐1 pathway. Thus, the potent inflammasome‐activating effect of (‐)‐maackiain may be clinically useful as an acute immune‐stimulating agent., Proinflammatory cytokine interleukin‐1β (IL‐1β) plays critical roles in defending against infections. Sophora flavescens‐derived (‐)‐maackiain potently amplifies caspase‐1 cleavage in response to nigericin. Notably, (‐)‐maackiain promotes caspase‐1 cleavage and IL‐1β production even in monophosphoryl lipid A‐primed macrophages. The potent inflammasome‐activating effect of (‐)‐maackiain may be clinically useful as an acute immune‐stimulating agent.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: i) abnormal lipid metabolism causing a delayed metabolic reprogramming and ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

The complete blood count (CBC) is one of the most important clinical steps in clinical diagnosis. The instruments used for CBC are usually expensive and bulky and require well-trained operators. Therefore, it is difficult for medical institutions below the tertiary level to provide these instruments, especially in underprivileged countries. Several reported on-chip blood cell tests are still in their infancy and do not deviate from conventional microscopic or impedance measurement methods. In this study, we (i) combined magnetically activated cell sorting and the differential density method to develop a method to selectively isolate three types of leukocytes from blood and obtain samples with high purity and concentration for portable leukocyte classification using the lens-free shadow imaging technique (LSIT), and (ii) established several shadow parameters to identify the type of leukocytes in a complete leukocyte shadow image by shadow image analysis. The purity of the separated leukocytes was confirmed by flow cytometry. Several shadow parameters such as the “order ratio” and “minimum ratio” were developed to classify the three types of leukocytes. A shadow image library corresponding to each type of leukocyte was created from the tested samples. Compared with clinical reference data, a correlation index of 0.98 was obtained with an average error of 6% and a confidence level of 95%. This technique offers great potential for biological, pharmaceutical, environmental, and clinical applications, especially where point-of-care detection of rare cells is required.

The value-added products in livestock industry is one of the key issues in order to maximize the revenue and to create a new business model. Numerous studies have suggested application of herbal plants as feed additives to increase health, productivity, and/or high-quality product in livestock. In this study, the first experiment was designed to develop in vitro evaluation system by using primary chicken myoblast (pCM) cells isolated from pectoralis major of 10-day-old male embryos. Subsequently, to evaluate effects of Korean Danggui Angelica gigas Nakai (AGN), we optimized the concentration of AGN root extract for treatment of primary pCM cells. After the treatment of AGN root extract, we compared proliferation and differentiation capacity, and also examined the gene expression. In the second experiment, the next generation sequencing analysis was performed to compare the different patterns of the global gene expression in pCM cells treated with AGN extract. Three up-regulated (pancreas beta cells, fatty acid metabolism and glycolysis) and one down-regulated (adipogenesis) gene sets were characterized suggesting that the AGN extract affected the metabolic pathways for the utilization of fat and glucose in chicken muscle cells. Furthermore, we validated the expression patterns of the up-regulated genes (GCLC, PTPN6, ISL1, SLC25A13, TGFBI, and YWHAH) in the AGN-treated pCM cells by quantitative RT-PCR. These results demonstrated that the treatment of AGN extract decreased proliferation and differentiation of pCM cells, and affected the metabolic pathways of glucose and fatty acids. Moreover, AGN extract derived from byproducts such as stem and leaf also showed the reduced proliferation patterns on AGN-treated pCM cells. Taken together, pCM cell-based in vitro assay system could be primarily and efficiently applied for evaluating the biofunctional efficacy of various feed additive candidates.

Cancer is caused by uncontrolled cell division and is a leading cause of mortality worldwide.

Glycogen storage disease type Ib (GSD-Ib), caused by a deficiency in glucose-6-phosphate transporter (G6PT), is characterized by disrupted glucose homeostasis, inflammatory bowel disease, neutropenia, and neutrophil dysfunction. The purpose of this study was to investigate the role of G6PT on macrophage functions and metabolism. Peritoneal macrophages of G6pt-/- mice were lower in number and their effector functions including migration, superoxide production, and phagocytosis were impaired. To investigate the underlying mechanisms of macrophage dysfunction, the G6PT gene was mutated in porcine alveolar macrophage 3D4/31 cells using the CRISPR/Cas9 technology. The G6PT-deficient macrophages exhibited significant decline in cell growth, bactericidal activity, and antiviral response. These phenotypes are associated with the impaired glycolysis and mitochondrial oxidative phosphorylation. We therefore propose that the G6PT-mediated metabolism is essential for effector functions of macrophage, the immune deficiencies observed in GSD-Ib extend beyond neutropenia and neutrophil dysfunction, and future therapeutic targets aimed both the neutrophils and macrophages may be necessary.

Ferritin (FT), a cage-protein composed of 24 subunits, may carry small molecules inside the core of cage. To enhance the potential of FT as a more functional drug carrier, FT genetically-fused with silaffin R5 peptide at N-terminus (R5FT) and silica-enveloped cage protein (SiO2/R5FT) were developed. Doxorubicin (Dox) can be loaded into cage interior by re-assembly of R5FT. The release of Dox loaded in internal cages was retarded by the silica-coated matrix and showed the pH-controlled behavior, in which the release amount is larger in acidic environments than neutral. R5FT provides a biocompatible and eco-friendly manufacturing process for the drug-laden silica nanoparticles over conventional production methods.

Hafnium-aluminum zinc oxide (HAZO) films as an active layer of oxide-transistors with different hafnium (Hf) contents were deposited via co-sputtering of separate targets. The effects of the sputtering power during co-sputtering on the structural, optical, electrical, and chemical properties of the HAZO films were examined. As the sputtering power increased, the structure of the HAZO films changed from polycrystalline to amorphous, and the Hf O bonds in the HAZO films increased, but the Zn O bonds decreased. Also, a bottom-gate-type thin-film transistor (TFT) using the HAZO film as its channel layer was fabricated and characterized. The TFTs using HAZO layer at room temperature as channel layer exhibited the device characteristics, such as a field effect mobility of 0.45 cm2/V·s, a threshold voltage of 17.18 V, a subthreshold swing of 0.85 V/decade, an on/off current ratio of 3.68 × 107, and a visible transmittance of 82.7%. It was discovered that the changes of the electrical characteristics of the HAZO TFTs were closely related to the changes of the Zn O/Hf O bonding ratio.

Cellular metabolism generally refers to biochemical processes that produce or consume energy within the cell. Recent studies have established that aberrant metabolic states caused by internal or external stresses and genetic mutations are intertwined with several human pathologies. Gaining insight into these metabolic alterations is, therefore, essential for understanding the pathophysiology of various diseases. Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by hypoglycemia, excessive glycogen accumulation in the liver and kidney, neutropenia, neutrophil dysfunction, and inflammatory bowel disease. GSD-Ib is caused by a deficiency of glucose-6-phosphate transporter (G6PT). Recently, it was reported that deficiency of G6PT also leads to the aberrant proliferation and differentiation of mesenchymal stem cells and impaired regulatory T-cell function. This review describes the broad impact of altered cellular metabolism resulting from a lack of G6PT activity on cellular function and considers the prospects of developing novel approaches for GSD-Ib treatment.

The vine stem of Spatholobus suberectus Dunn (SS) is used as a traditional herbal medicine in China. Chinese herbal medicines are well known as natural bioactive compounds that can be used as new medicines, and their antioxidant and anticancer effects have also been reported. This study aimed to examine the anticancer effect of a high-pressure hot-water SS extract on rat C6 glioma cells. The SS extract effectively suppressed the viability and proliferation of C6 glioma cells through an antioxidant effect. Reactive oxygen species (ROS) levels in cancer cells are higher than that in normal cells. If the ROS level falls below that required for the growth of cancer cells, their rapid proliferation and growth can be suppressed. We also measured the induction of mitochondrial membrane depolarization and cell cycle arrest effect caused by the SS extract in C6 glioma cells through a FACS analysis. In addition, we observed an increase in STAT3, p53, E2F1, and p21 mRNA expression and a decrease in Bcl-2 mRNA expression by quantitative PCR. An increase in p21 protein expression of over 83% was observed through western blot analysis. All these data support the fact that the high-pressure hot-water SS extract has the potential to be used for glioma treatment.

Glycogen storage disease type Ib (GSD-Ib) is caused by mutations of the glucose-6-phosphate transporter (G6PT) and characterized by disrupted glucose homeostasis, neutropenia, and neutrophil dysfunction. To investigate the role of G6PT in human adipose-derived mesenchymal stem cells (hMSCs), the G6PT gene was mutated by CRISPR/Cas9 technology and single cell-derived G6PT-/- hMSCs were established. G6PT-/- hMSCs have significantly increased cell proliferation but impaired adipogenesis and osteogenesis. These phenotypes are associated with two mechanisms: i) metabolic reprogramming in G6PT-/- hMSCs causing a metabolic shift toward glycolysis rather than oxidative phosphorylation and ii) increased cyclooxygenase-2-derived prostaglandin E2 secretion in G6PT-/- hMSCs. This study demonstrates that G6PT is essential for proliferation and differentiation of MSCs, providing important insights into the GSD-Ib phenotypes.

Gliomas are one of the most common types of primary brain tumors, characterized by rapid proliferation and infiltration into normal brain tissue. Corncob is the most plentiful byproducts of Zea mays L., of which anti-cancer effect has not been reported. Therefore, we aimed to examine the anti-proliferative effect of a high-pressure hot-water extract of corncob on glioma cells and elucidated the underlying mechanism. The high-pressure hot-water corncob extract contained approximately 94.8 mg/g and 1.82 μg/g of total phenol and catechin, respectively. Glioma cell treated with different concentrations of high-pressure hot-water corncob extract was shown to be suppressed in growth during three days of culture. In parallel, corncob extract reduced the glioma cell viability and induced cell cycle arrest in G0/G1 phase by upregulating the expression level of cyclin-dependent kinase inhibitor p21. Decreased proliferation and viability in glioma cells treated with corncob extract can be attributed to reduced reactive oxygen species (ROS), antiapoptotic Bcl-2 protein, and a lactate transporter monocarboxylate transporter 1 of which levels are higher than those in normal cells. Based on its inhibitory effects on proliferation and viability of C6 glioma cells, a high-pressure hot-water corncob extract has the potential to be used for glioma treatment.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt(−/−)) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt(−/−) mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt(−/−)mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the β2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of β2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

The SLC37 family consists of four sugar-phosphate exchangers, A1, A2, A3, and A4, which are anchored in the endoplasmic reticulum (ER) membrane. The best characterized family member is SLC37A4, better known as the glucose-6-phosphate (G6P) transporter (G6PT). SLC37A1, SLC37A2, and G6PT function as phosphate (Pi)-linked G6P antiporters catalyzing G6P:Pi and Pi:Pi exchanges. The activity of SLC37A3 is unknown. G6PT translocates G6P from the cytoplasm into the lumen of the ER where it couples with either glucose-6-phosphatase-α (G6Pase-α) or G6Pase-β to hydrolyze intraluminal G6P to glucose and Pi. The functional coupling of G6PT with G6Pase-α maintains interprandial glucose homeostasis and the functional coupling of G6PT with G6Pase-β maintains neutrophil energy homeostasis and functionality. A deficiency in G6PT causes glycogen storage disease type Ib, an autosomal recessive disorder characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. Neither SLC37A1 nor SLC37A2 can functionally couple with G6Pase-α or G6Pase-β, and there are no known disease associations for them or SLC37A3. Since only G6PT matches the characteristics of the physiological ER G6P transporter involved in blood glucose homeostasis and neutrophil energy metabolism, the biological roles for the other SLC37 proteins remain to be determined.

Pravastatin is a lipid-lowering agent that attenuates atherosclerosis. However, the multifactorial pathogenesis of atherosclerosis requires other drugs with different anti-atherogenic mechanisms. We chose sarpogrelate as an anti-platelet agent and a novel component of a complex drug with pravastatin due to its high potential but little information on its beneficial effects on atherosclerosis. Low-density lipoprotein receptor-knockout mice were fed a high-fat, high-cholesterol diet and treated with pravastatin alone, sarpogrelate alone, or a combination of both drugs. Although sarpogrelate alone did not significantly reduce atherosclerotic plaque areas, co-treatment with pravastatin significantly decreased aortic lesions compared to those of the pravastatin alone treated group. The combined therapy was markedly more effective than that of the single therapies in terms of foam cell formation, smooth muscle cell proliferation, and inflammatory cytokine levels. These results suggest that pravastatin and sarpogrelate combined therapy may provide a new therapeutic strategy for treating atherosclerosis.

Glycogen storage disease type Ia (GSD-Ia) patients, deficient in glucose-6-phosphatase-alpha, manifest disturbed glucose homeostasis with long-term renal disease. We have previously shown that renal fibrosis in GSD-Ia is mediated by the angiotensin/transforming growth factor-beta1 (TGF-beta1) pathway, which also elicits renal damage through oxidative stress. In this study, we further elucidate the mechanism of renal disease by showing that renal expression of Nox-2, p22(phox), and p47(phox), components of NADPH oxidase, are upregulated in GSD-Ia mice compared with controls. Akt/protein kinase B, a downstream mediator of angiotensin II and TGF-beta1, is also activated, leading to phosphorylation and inactivation of the Forkhead box O family of transcription factors. This in turn triggers downregulation of superoxide dismutase and catalase (CAT) activities that have essential roles in oxidative detoxification in mammals. Renal oxidative stress in GSD-Ia mice is shown by increased oxidation of dihydroethidium and by oxidative damage of DNA. Importantly, renal dysfunction, reflected by elevated serum levels of blood urea nitrogen, reduced renal CAT activity, and increased renal fibrosis, is improved in GSD-Ia mice treated with the antioxidant drug tempol. These data provide the first evidence that oxidative stress is one mechanism that underlies GSD-Ia nephropathy.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that works with a liver/kidney/intestine–restricted glucose-6-phosphatase-α (G6Pase-α) to maintain glucose homeostasis between meals. Clinically, GSD-Ib patients manifest disturbed glucose homeostasis and neutrophil dysfunctions but the cause of the latter is unclear. Neutrophils express the ubiquitously expressed G6PT and G6Pase-β that together transport G6P into the endoplasmic reticulum (ER) lumen and hydrolyze it to glucose. Because we expected G6PT-deficient neutrophils to be unable to produce endogenous glucose, we hypothesized this would lead to ER stress and increased apoptosis. Using GSD-Ib mice, we showed that GSD-Ib neutrophils exhibited increased production of ER chaperones and oxidative stress, consistent with ER stress, increased annexin V binding and caspase-3 activation, consistent with an increased rate of apoptosis. Bax activation, mitochondrial release of proapoptotic effectors, and caspase-9 activation demonstrated the involvement of the intrinsic mitochondrial pathway in these processes. The results demonstrate that G6P translocation and hydrolysis are required for normal neutrophil functions and support the hypothesis that neutrophil dysfunction in GSD-Ib is due, at least in part, to ER stress and increased apoptosis.

Comparative analysis of binding of intact glucose-grownFibrobacter succinogenesstrain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grownF. succinogeneswere identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system ofF. succinogenes.

Human health problems due to long life are becoming major issues in society, and in particular greater interest collected on women's health after menopause. Many substances can be introduced to women's health, however, materials from the substances have not shown all of the safety and efficacy properties that are not easily found. Currently, it is known about the effects of the disease on the female insect-derived material that is capable of overcoming this problem significantly. When using the insect-derived material through the results of several studies suggest that it is possible to solve a hormonal imbalance and nutritional imbalance in the elderly. Here, we'd like to try to dissertate about the new trends for women's health improvement using novel materials-derived from insects.

Batten disease (BD; also known as juvenile neuronal ceroid lipofuscinosis) is a genetic disorder inherited as an autosomal recessive trait and is characterized by blindness, seizures, cognitive decline, and early death resulting from the inherited mutation of the CLN3 gene. Mitochondrial oxidative stress, endoplasmic reticulum (ER) stress, disrupted autophagy, and enhanced apoptosis have been suggested to play a role in BD pathogenesis. Fibrates, a class of lipid-lowering drugs that induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, are the most commonly used PPAR agonists. Assuming that fibrates have a neuroprotective effect, we studied the effects of fibrates, fenofibrate, bezafibrate, and gemfibrozil on apoptosis, depolarization of mitochondrial membrane, and defective autophagy in BD lymphoblast cells. The viability of fibrate-treated BD lymphoblast cells increased to levels of normal lymphoblast cells. In addition, treatment with fibrates inhibited depolarization of mitochondrial membrane potential in BD lymphoblast cells. Defective autophagy in BD lymphoblast cells was normalized when treated with fibrates as indicated by increased acridine orange staining. The recovery of autophagy in BD lymphoblast cells is most likely attributed to the upregulation of autophagy proteins, lysosomal-associated membrane protein 1 (LAMP1), and LC3 I/II, after treatment with fibrates. This study therefore suggests that fibrates may have a therapeutic potential against BD.

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively. The CDs of the two esterases exhibited the highest similarity to CDs of acetylxylan esterases: AxeA from the ruminal fungi Orpinomyces sp. and BnaA from Neocallimastix patriciarum. Axe6A and Axe6B were optimally active at neutral pH and had low Kmvalues of 0.084 and 0.056 mmol·L–1, respectively. Axe6A and Axe6B were shown to bind to insoluble cellulose and xylan and to soluble arabinoxylan. Axe6A deacetylated acetylated xylan at the same initial rate in the presence and absence of added Xyn10E xylanase from F. succinogenes, but the action of the xylanase on acetylated xylan was dependent upon the initial activity of Axe6A. The capacity of acetylxylan esterases to bind to plant cell wall polymers and to independently deacetylate xylan enabling xylanase to release xylooligo saccharides, documents the central role these enzymes have to improve access of F. succinogenes to cellulose.Key words: Fibrobacter succinogenes S85, acetylxylan esterase, xylanase, synergy.

Neutrophils play an essential role in defense against intruding microorganisms. They are produced in great number in the bone marrow and circulate in blood where they are found in a quiescent state. In response to inflammatory stimuli, neutrophils take different steps-rolling, adhesion, and transmigration-to migrate towards inflammation sites. In this study, we investigated the underlying mechanisms of impaired neutrophil adhesion in glycogen storage disease type Ib (GSD-Ib) which is caused by a deficiency of the glucose-6-phosphate transporter (G6PT). GSD-Ib is characterized not only by disrupted glucose homeostasis but also by neutropenia and neutrophil dysfunction. GSD-Ib mice were infused with a recombinant adeno-associated virus (rAAV) vector expressing human G6PT only in liver to increase their survival. Unlike control mice, GSD-Ib-rAAV mice manifested severe neutropenia in both blood and bone marrow and neutrophils of GSD-Ib-rAAV mice were defective in adhesion to tumor necrosis factor-a-treated epithelial cells and intercellular adhesion molecule 1 and fibrinogen. The β2-integrins including CD11a/CD18 and CD11b/CD18 are recognized as vital players in neutrophil recruitment. Consistent with impaired neutrophil adhesion, the expression of CD11a and CD11b were found to be decreased in neutrophils of GSD-Ib-rAAV mice, compared with that of control mice. Particularly neutrophils of GSD-Ib-rAAV mice showed increased proteolytic degradation of CD11b. Given the central role of β2-integrins in neutrophil adhesion, this alternation of CD11a and CD11b molecules and their effect on neutrophil adhesion probably define a molecular mechanism to neutrophil dysfunction manifested in GSD-Ib.

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency, also known as severe congenital neutropenia syndrome 4, is characterized not only by neutropenia but also by impaired neutrophil energy homeostasis and functionality. We now show the syndrome is also associated with macrophage dysfunction, with murine G6pc3−/− macrophages having impairments in their respiratory burst, chemotaxis, calcium flux, and phagocytic activities. Consistent with a glucose-6-phosphate (G6P) metabolism deficiency, G6pc3−/− macrophages also have a lower glucose uptake and lower levels of G6P, lactate, and ATP than wild-type macrophages. Furthermore, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated, and G6pc3−/− macrophages exhibit repressed trafficking in vivo both during an inflammatory response and in pregnancy. During pregnancy, the absence of G6Pase-β activity also leads to impaired energy homeostasis in the uterus and reduced fertility of G6pc3−/− mothers. Together these results show that immune deficiencies in this congenital neutropenia syndrome extend beyond neutrophil dysfunction.

Glycogen storage disease type Ia (GSD-Ia), which is characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by deficiencies in the endoplasmic reticulum (ER)-associated glucose-6-phosphatase-α (G6Pase-α or G6PC) that hydrolyzes glucose-6-phosphate (G6P) to glucose. G6Pase-α activity depends on the G6P transporter (G6PT) that translocates G6P from the cytoplasm into the ER lumen. The functional coupling of G6Pase-α and G6PT maintains interprandial glucose homeostasis. We have shown previously that gene therapy mediated by AAV-GPE, an adeno-associated virus (AAV) vector expressing G6Pase-α directed by the human G6PC promoter/enhancer (GPE), completely normalizes hepatic G6Pase-α deficiency in GSD-Ia (G6pc−/−) mice for at least 24 weeks. However, a recent study showed that within 78 weeks of gene deletion, all mice lacking G6Pase-α in the liver develop HCA. We now show that gene therapy mediated by AAV-GPE maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild-type hepatic G6Pase-α activity. The treated mice displayed normal hepatic fat storage, had normal blood metabolite and glucose tolerance profiles, had reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast, and had no evidence of hepatic abnormalities. After a 24-hour fast, hepatic G6PT messenger RNA levels in G6pc−/− mice receiving gene therapy were markedly increased. Because G6PT transport is the rate-limiting step in microsomal G6P metabolism, this may explain why the treated G6pc−/− mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. Conclusion: These results confirm that AAV-GPE–mediated gene transfer corrects hepatic G6Pase-α deficiency in murine GSD-Ia and prevents chronic HCA formation. (HEPATOLOGY 2012;56:1719–1729)

G6PC3 (or glucose-6-phosphatase-β) deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced endoplasmic reticulum (ER) stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Here we show that murine G6pc3−/− neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate/Akt signaling pathways, and that neutrophil apoptosis is mediated in part by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony-stimulating factor (G-CSF) improves neutropenia, but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3−/− neutrophils in culture exhibit accelerated apoptosis compared with wild-type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6mM) accelerates apoptosis but is more pronounced for wild-type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, phosphatidylinositol 3,4,5-trisphosphate, and active caspase-3. Neutrophils from in vivo G-CSF–treated G6pc3−/− mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and adenosine-5′-triphosphate, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3−/− neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis.

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes , Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

Women's health has been threatened by various diseases mainly including heart disease, breast cancer, osteoporosis, depression, and autoimmune disease. But development of medication for these diseases has been restricted by high development costs and low success rates. Herein the attempt to develop valid bioactive materials from a traditional natural material has been made. Resveratrol has been reported to be effective in treatment of breast cancer and heart disease. Goji berry has received attention as a natural based therapeutic material to treat a diabetes, cardiovascular disease, and osteoporosis. Leonurus family has been reported to be effective particularly in pregnant women due to high contents of vitamin as well as stimulation of uterine contraction. Annona family has effects such as anti-anxiety, anticonvulsant and recently it is proposed to be as a therapeutic material to cure depression based on its strong antidepressant effect. Shiraia bambusicola has been utilized to cure angiogenesis-related disease from ancient China and furthermore recently it was proved to be effective in rheumatoid arthritis. Getting an understanding of utilization of these traditional natural materials not only enhances the interest in development of therapeutic materials for preventing and treating various women's diseases, but also makes it possible to develop novel therapeutic materials.

Neutropenia and neutrophil dysfunction are common in many diseases, although their etiology is often unclear. Previous views held that there was a single ER enzyme, glucose-6-phosphatase-alpha (G6Pase-alpha), whose activity--limited to the liver, kidney, and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis, in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently, we characterized a second G6Pase activity, that of G6Pase-beta (also known as G6PC), which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst, chemotaxis, and calcium flux; and increased susceptibility to bacterial infection. Consistent with this, G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow, and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology, providing a potential model for the treatment of these conditions.

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) is a ubiquitously expressed glucose-6-phosphate (G6P) hydrolase that catalyzes the hydrolysis of G6P to glucose and phosphate. It is a key enzyme for intracellular glucose production in non-gluconeogenic organs. Mutations in G6Pase-β underlie severe congenital neutropenia syndrome type 4 (SCN4, MIM612541), an autosomal recessive disease characterized by neutropenia and neutrophil dysfunction. A mouse model for G6Pase-β deficiency (G6pc3-/-) established by gene targeting manifests both neutropenia and neutrophil dysfunction, mimicking the human disorder. Recent studies have shown that in G6Pase-β deficiency enhanced neutrophil apoptosis leads to neutropenia while disrupted energy homeostasis underlies neutrophil dysfunction. The patients of G6Pase-β deficiency also present with non-haematological defects, including prominent superficial venous pattern, congenital cardiac anomaly, myopathy, disrupted bone remodeling, and genital anomalies, suggesting that G6Pase-β deficiency leads to a broader cell dysfunction. We now hypothesize that these non-haematological defects may reflect loss of glucose metabolism within mesenchymal stem cells (MSC) that require levels of glucose beyond those supplied by the blood. MSC are multipotent stem cells that are capable of differentiating into all connective tissue types including bone, cartilage, myocytes, and adipocytes. Studies have shown that MSC cultured in reduced glucose concentrations exhibit increased proliferative ability and reduced replicative senescence. Using MSC isolated from the compact bones, we show that G6pc3-/- MSC grow at a significantly faster rate than those of wild type MSC under both normoxia and hypoxia conditions. Consistently, the colony-forming unit-fibroblastic counts in G6pc3-/- MSC are 2-fold higher than those in wild type MSC, suggesting that G6pc3-/- MSC contain higher numbers of progenitors. Differentiation of MSC involves metabolic shifts between glycolysis and mitochondrial oxidative phosphorylation. An increase in the ratio of lactate to pyruvate correlates with increased glycolysis and an increase in oxygen consumption measures increased mitochondrial oxidative phosphorylation. Adipogenesis and osteogenesis of MSC have been shown to be associated with increased oxygen consumption, while chondrogenesis of MSC is associated with increased glycolysis. We now show that cellular levels of lactate and ATP in G6pc3-/- MSC are 4.5- and 2-fold higher, respectively than those in wide type MSC. Moreover, G6pc3-/- MSC exhibit a 2.5-fold increase in the ratio of lactate to pyruvate but a decrease in oxygen consumption, compared to wild type MSC, suggesting that G6Pase-β-deficient MSC exhibit enhanced glycolysis along with impaired mitochondrial respiration activity. This also suggests that adipogenesis and osteogenesis of MSC may be impaired in G6pc3-/- MSC. Adipogenic differentiation of MSC from mice was then evaluated by morphological alterations and lipid accumulation. MSC from wild type mice, cultured for 3 days in adipogenic differentiation medium, changed from fibroblast-like cells to round-shaped adipocyte-like cells, while G6pc3-/- MSC cultured under the same conditions did not show any morphological changes. Wild type MSC, cultured for 5 days in adipogenic differentiation medium, yielded many adipocytes containing lipid droplets and staining positive by Oil red O, while adipocytes were rarely seen in G6pc3-/- MSC cultured under similar conditions, confirming the impaired adipogenic differentiation of G6pc3-/- MSC. In conclusion, the non-hematological defects associated with G6Pase-β deficiency may result from altered energy homeostasis in G6pc3-/- MSC, leading to enhanced glycolysis along with impaired mitochondrial respiration activity, that impacts MSC differentiation. Disclosures No relevant conflicts of interest to declare.

Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER) of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate (P(i)). This reaction depends on coupling the G6P transporter (G6PT) with glucose-6-phosphatase-α (G6Pase-α). Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i)-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i) exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i)-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.