Effect of dipeptidyl peptidase IV inhibitors, thiazolidinedione, and sulfonylurea on osteoporosis in patients with type 2 diabetes: population-based cohort study.

The population-based cohort study used the Korean National Health Insurance claims database to evaluate the effect of anti-diabetic drugs on osteoporosis. The use of DPP-IV inhibitors does not increase the risk of osteoporosis compared with the use of sulfonylureas in patients with type 2 diabetes mellitus, while a weak association was found between thiazolidinediones and increased risk of osteoporosis. PURPOSE: The current study aimed to evaluate the effect of dipeptidyl peptidase IV inhibitors (DPP-IVi), thiazolidinedione (TZD), and sulfonylurea (SU) on osteoporosis in patients with type 2 diabetes. METHODS: A population-based cohort study was conducted in the Republic of Korea using the Korean National Health Insurance claims database. Data from 2012 to 2017 for patients of 50-99 years of age who were prescribed DPP-IVi, TZD, or SU during 2013-2015 were extracted from the database. Based on pre-defined criteria, a total of 381,404 patients were analyzed after inverse probability of treatment weighting. The association between the study drugs and osteoporosis was estimated using Cox proportional hazards models. Data of 220,166 patients who were prescribed DPP-IVi, 18,630 who were prescribed TZD, and 142,608 patients who were prescribed SU were set. RESULTS: In the multivariate-adjusted analysis, the hazard ratio (HR) of osteoporosis in the DPP-IVi group was not significantly different from that of the SU group (HR: 0.97; 95% confidence interval (CI) 0.94-1.00), whereas the HR of osteoporosis in the TZD group was higher (HR: 1.13; 95% CI 1.06-1.20). In the subgroup analysis, the HRs of osteoporosis were higher with pioglitazone (HR: 1.14; 95% CI 1.06-1.23) in the TZD group and with glibenclamides (HR: 1.39; 95% CI 1.09-1.77) in the SU group, whereas drugs with lower HR in the DPP-IVi group were saxagliptin (HR: 0.93; 95% CI 0.87-0.99) and sitagliptin (HR: 0.93; 95% CI 0.89-0.97). CONCLUSION: DPP-IV inhibitors do not increase the risk of osteoporosis compared with sulfonylureas in patients with type 2 diabetes mellitus, while a weak association was found between thiazolidinediones and increased risk of osteoporosis.

The utility of ultrasound superb microvascular imaging for evaluation of breast tumour vascularity: comparison with colour and power Doppler imaging regarding diagnostic performance.

AIM: To investigate the utility of superb microvascular imaging (SMI) for evaluating the vascularity of breast masses in comparison with colour or power Doppler ultrasound (US) and the effect on diagnostic performance. MATERIALS AND METHODS: A total of 191 biopsy-proven masses (99 benign and 92 malignant) in 166 women with greyscale, colour Doppler, power Doppler, and SMI images were enrolled in this retrospective study. Three radiologists analysed the vascular images using a three-factor scoring system to evaluate the number, morphology, and distribution of tumour vessels. They assessed the Breast Imaging-Reporting and Data System categories for greyscale US alone and combinations of greyscale US and each type of vascular US. The Kruskal-Wallis test was performed and the area under the receiver-operating characteristic curve (AUC) measured. On SMI, vascular scores were compared between benign and malignant masses and the optimal cut-off value for the overall score was determined. RESULTS: SMI showed higher vascular scores than colour or power Doppler US and malignant masses had higher scores than benign masses (p<0.001). The diagnostic performance of the combination of greyscale US and SMI was higher than those of greyscale US alone and greyscale and colour or power Doppler US (AUC, 0.815 versus 0.774, 0.789, 0.791; p<0.001). The optimal cut-off value of the overall vascular score was 5 with a sensitivity of 82.3% and a specificity of 65.3% (AUC, 0.808). CONCLUSION: SMI is superior to colour or power Doppler US for characterising the vascularity in breast masses and improving diagnostic performance.

Effects of serial passage on the characteristics and chondrogenic differentiation of canine umbilical cord matrix derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

Identifying different types of microorganisms with terahertz spectroscopy.

Most microbial detection techniques require pretreatment, such as fluorescent labeling and cultivation processes. Here, we propose novel tools for classifying and identifying microorganisms such as molds, yeasts, and bacteria based on their intrinsic dielectric constants in the THz frequency range. We first measured the dielectric constant of films that consisted of a wide range of microbial species, and extracted the values for the individual microbes using the effective medium theory. The dielectric constant of the molds was 1.24-1.85, which was lower than that of bacteria ranging from 2.75-4.11. The yeasts exhibited particularly high dielectric constants reaching 5.63-5.97, which were even higher than that of water. These values were consistent with the results of low-density measurements in an aqueous environment using microfluidic metamaterials. In particular, a blue shift in the metamaterial resonance occurred for molds and bacteria, whereas the molds have higher contrast relative to bacteria in the aqueous environment. By contrast, the deposition of the yeasts induced a red shift because their dielectric constant was higher than that of water. Finally, we measured the dielectric constants of peptidoglycan and polysaccharides such as chitin, α-glucan, and ß-glucans (with short and long branches), and confirmed that cell wall composition was the main cause of the observed differences in dielectric constants for different types of microorganisms.

Regulation of food intake and energy expenditure by hypothalamic malonyl-CoA.

Energy balance is monitored by the hypothalamus. Malonyl-CoA, an intermediate in fatty acid synthesis, serves as an indicator of energy status in the hypothalamic neurons. The cellular malonyl-CoA level is determined by its rate of synthesis, catalyzed by acetyl-CoA carboxylase (ACC), and rate of removal, by fatty acid synthase (FAS). Malonyl-CoA functions in the hypothalamic neurons that express orexigenic and anorexigenic neuropeptides. Inhibitors of FAS, administered systemically or intracerebroventricularly to mice, increase hypothalamic malony-CoA and suppress food intake. Recent evidence suggests that the changes of hypothalamic malonyl-CoA during feeding and fasting cycles are caused by changes in the phosphorylation state and activity of ACC mediated via 5'-AMP-activated protein kinase (AMPK). Stereotactic delivery of a viral malonyl-CoA decarboxylase (MCD) vector into the ventral hypothalamus lowers malonyl-CoA and increases food intake. Fasting decreases hypothalamic malonyl-CoA and refeeding increases hypothalamic malonyl-CoA, to alter feeding behavior in the predicted manner. Malonyl-CoA level is under the control of AMP kinase which phosphorylates/inactivates ACC. Malonyl-CoA is an inhibitor of carnitine palmitoyl-CoA transferase-1 (CPT1), an outer mitochondrial membrane enzyme that regulates entry into, and oxidation of fatty acids, by mitochondria. CPT1c, a recently discovered, brain-specific enzyme expressed in the hypothalamus, has high sequence similarity to liver/muscle CPT1a/b and binds malonyl-CoA, but does not catalyze the prototypical reaction. This suggests that CPT1c has a unique function or activation mechanism. CPT1c knockout (KO) mice have lower food intake, weigh less and have less body fat, consistent with the role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c protects against the effects of a high-fat diet. CPT1cKO mice exhibit decreased rates of fatty acid oxidation, consistent with their increased susceptibility to diet-induced obesity. We suggest that CPT1c may be a downstream target of malonyl-CoA that regulates energy homeostasis.

Light-chain-restricted germinal centres in reactive lymphadenitis: report of eight cases.

AIMS: Light-chain-restricted germinal centres are generally associated with the existence of a neoplastic lymphoproliferative disorder. The aim was to present a series of cases with persistent lymph node enlargement that featured some germinal centres showing light chain immunoglobulin restriction. METHODS AND RESULTS: A series of six reactive lymphadenitis and two Castleman's disease cases was analysed by immunohistochemistry, IgH-polymerase chain reaction (PCR) and microdissected PCR. In all cases some germinal centres contained a population of plasma cells and plasmacytoid germinal centre cells showing light chain immunoglobulin restriction. In three cases the monotypic cells also showed distinct Bcl-2 expression. Two of the cases showed a predominant IgH rearrangement on a florid polyclonal background and one had an IgH monoclonal rearrangement, as revealed by PCR. Microdissected germinal centre PCR revealed a dominant repeated band in one of three cases and in another case a non-repeated clonal peak was observed. One of the patients developed a follicular lymphoma, which became evident from a subsequent biopsy. CONCLUSIONS: These findings may be a manifestation of an underlying disorder in the regulation of the immune response, or an exaggeration of the germinal centre oligoclonal nature. This should be taken into account in the differential diagnosis of follicular hyperplasia.

Secondary amyloidosis associated with histiocytosis X.

We report a 34-year-old man diagnosed with Langerhans cell histiocytosis (LCH) or histiocytosis X in 1980. He had multiple focal osseous lesions, difficult control of the disease activity and was treated many times with chemo- and radiotherapy for symptomatic control. His kidney disease started 20 years after the diagnosis with progressive renal failure and increasing non-nephrotic proteinuria, coinciding with two flares of LCH. A percutaneous renal biopsy demonstrated amyloidosis. There is only one case described in the amyloidosis literature associated with LCH.

Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection.

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n=10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n=10) were IM inoculated with minimum essential medium (MEM). At approximately 2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC=0.97), the N ELISA (AUC=0.96), and the M 3' ELISA (AUC=0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes was a poor prognosticator of PRRSV infection status.

Enhanced sensitivity in THz plasmonic sensors with silver nanowires.

We developed hybrid slot antenna structures for microbial sensing in the THz frequency range, where silver nanowires (AgNWs) were employed to increase the sensitivity. In order to fabricate the hybrid devices, we partially etched the AgNW in the slot antenna region, where we can expect the field enhancement effect at the AgNW tip. We measured the resonant-frequency shift observed upon the deposition of a polymer layer, and observed that the sensitivity increased upon the introduction of AgNWs, with an enhancement factor of more than four times (approximately six times in terms of figure-of-merit). The sensitivity increased with the AgNW density until saturation. In addition, we tested devices with PRD1 viruses, and obtained an enhancement factor of 3.4 for a slot antenna width of 3 µm. Furthermore, we performed finite-difference time-domain simulations, which confirmed the experimental results. The sensitivity enhancement factor decreased with the decrease of the slot width, consistent with the experimental findings. Two-dimensional mapping of the electric field confirmed the strong field localization and enhancement at the AgNW tips.

Production of mycelia and exo-biopolymer from molasses by Cordyceps sinensis 16 in submerged culture.

The molecular weight of exo-biopolymer obtained from a submerged culture of Cordyceps sinensis 16 consisted of a main unit and a subunit of 126 and 68 kDa, respectively. The optimal medium for the production of mycelia and exo-biopolymer was determined to be molasses containing 2% sucrose, 0.9% yeast extract, 0.3% K2HPO4, and 0.4% CaCl2. Using optimized medium, maximum productions of mycelia and exo-biopolymer in shake-flask culture were 54.0 g/L and 28.4 g/L, respectively. This study suggests that large-scale production of mycelia and exo-biopolymer by C. sinensis 16 is possible in submerged culture.

Small cell neuroendocrine carcinoma of the sinonasal region. A propose of a case.

We describe a 70-year-old man with rheumatoid arthritis and pulmonary fibrosis who presented with a month's history of pain in the left lateronasal region and inferior eyelid. On examination there was left exophthalmos, difficulty in coordinating eye movements, inflammation, erythema, and pain. Computed tomography showed a 3 cm mass in the left posterior ethmoid region, a biopsy specimen from which showed a small cell neuroendocrine tumour. He refused operation and was treated unsuccessfully with four cycles of cisplatin and etoposide.

Sensing viruses using terahertz nano-gap metamaterials.

We demonstrate highly sensitive detection of viruses using terahertz split-ring resonators with various capacitive gap widths. Two types of viruses, with sizes ranging from 60 nm (PRD1) to 30 nm (MS2), were detected at low densities on the metamaterial surface. The dielectric constants of the virus layers in the THz frequency range were first measured using thick films, and the large values found identified them as efficient target substances for dielectric sensing. We observed the resonance-frequency shift of the THz metamaterial following deposition of the viruses on the surface at low-density. The resonance shift was higher for the MS2 virus, which has a relatively large dielectric constant. The frequency shift increases with surface density until saturation and the sensitivity is then obtained from the initial slope. Significantly, the sensitivity increases by about 13 times as the gap width in the metamaterials is decreased from 3 µm to 200 nm. This results from a combination of size-related factors, leading to field enhancement accompanying strong field localization.

Deep tracheal laceration after balloon dilation for benign tracheobronchial stenosis: case reports of two patients.

We report two cases of deep tracheal laceration in female patients after balloon dilation for benign tracheobronchial stenosis. Immediate post-procedure bronchoscopy and CT including 3D reconstructions showed deep lacerations in the posterior tracheal wall. Clinically, the patients' dyspnoea subsided and there has been no recurrence during follow-up after balloon dilation. On the follow-up 3D-reconstructed CT scans obtained 2 months and 8 months following balloon dilation, respectively, the lacerations had healed completely and there was considerable improvement in lumen size.

Human cystine/glutamate transporter: cDNA cloning and upregulation by oxidative stress in glioma cells.

A human cDNA for amino acid transport system x(C)(-) was isolated from diethyl maleate-treated human glioma U87 cells. U87 cells expressed two variants of system x(C)(-) transporters hxCTa and hxCTb with altered C-terminus regions probably generated by the alternative splicing at 3'-ends. Both hxCTa and hxCTb messages were also detected in spinal cord, brain and pancreas, although the level of hxCTb expression appears to be lower than that of hxCTa in these tissues. When expressed in Xenopus oocytes, hxCTb required the heavy chain of 4F2 cell surface antigen (4F2hc) and exhibited the Na(+)-independent transport of L-cystine and L-glutamate, consistent with the properties of system x(C)(-). In agreement with this, 137 kDa band was detected by either anti-xCT or anti-4F2hc antibodies in the non-reducing condition in western blots, whereas it shifted to 50 kDa or 90 kDa bands in the reducing condition, indicating the association of two proteins via disulfide bands. We found that the expression of xCT was rapidly induced in U87 cells upon oxidative stress by diethyl maleate treatment, which was accompanied by the increase in the L-cystine uptake by U87 cells. Because of this highly regulated nature, xCT in glial cells would fulfill the task to protect neurons against oxidative stress by providing suitable amount of cystine to produce glutathione.

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines.

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation.

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

Identification of multispecific organic anion transporter 2 expressed predominantly in the liver.

In the present study, we demonstrate that NLT (novel liver-specific transport protein) is a multispecific organic anion transporter of the liver. The amino acid sequence of NLT shows 42% identity to that of the renal multispecific organic anion transporter, OAT1. When expressed in Xenopus laevis oocytes, NLT mediated uptake of organic anions, such as salicylate, acetylsalicylate, PGE2, dicarboxylates and p-aminohippurate. [14C]Salicylate uptake via NLT was saturable (Km = 88.8 +/- 23.4 microM) and sodium-independent. Expression of the mRNA of NLT was detected in the liver and kidney (liver >> kidney). We propose that NLT be renamed OAT2.

Sequence and transcription map analysis of early region-1 of porcine adenovirus type-3.

The nucleotide sequence of a region of the genome of porcine adenovirus-3 (PAV-3) between map units 1 and 12.2 was determined. The sequenced region included four major open reading frames, and several transcription control elements. Homology studies, using the deduced amino acid sequences of the open reading frames, revealed genes coding for the E1A, E1B 202R, E1B 474R and pIX proteins. The region was characterized by Northern blot analysis and sequencing of cDNA clones. In PAV-3, the E1A region is located between 1.5 and 3.8 map units. Alternate splice donor sites are used to produce four different types of transcripts from the primary transcript of the E1A region. The E1A proteins of PAV-3 contain a consensus zinc finger motif, which was shown to be the principal transactivation region of human adenovirus-5 (HAV-5) E1A proteins. The PAV-3 E1A proteins also contain a retinoblastoma susceptibility protein (pRb) binding motif, which in HAVs interacts with cellular Rb protein to overcome the pRb mediated transcription repression. The E1B region in PAV-3 maps between 4.0 and 12.2 map units, and shares a polyadenylation signal and polyadenylation sites with the gene coding for pIX. A single major and a number of minor mRNA species are produced from the E1B region. The open reading frame (ORF) analysis of cDNA representing major mRNA produced from the E1B region showed two overlapping ORFs corresponding to 19K and 55K ORFs of HAV-2. In PAV-3, the gene coding for pIX is located between 9.9 and 12.2 map units and codes for a protein of 199 amino acids.

Isolation of peptide ligands that inhibit glutamate racemase activity from a random phage display library.

Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing antibiotic substances. The new agents include compounds that interfere with bacterial membrane function. The peptidoglycan component of the bacterial cell wall is synthesized by glutamate racemase, and this enzyme is responsible for the biosynthesis of d-glutamate, which is an essential component of cell wall peptidoglycan. In this study, we screened a phage display library expressing random dodecapeptides on the surface of bacteriophage against an Escherichia coli glutamate racemase, and isolated specific peptide sequences that bind to the enzyme. Twenty-seven positive phage clones were analyzed, and seven different peptide sequences were obtained. Among them, the peptide sequence His-Pro-Trp-His-Lys-Lys-His-Pro-Asp-Arg-Lys-Thr was found most frequently, suggesting that this peptide might have the highest affinity to glutamate racemase. The positive phage clones and HPWHKKHPDRKT synthetic peptide were able to inhibit glutamate racemase activity in vitro, implying that our peptide inhibitors may be utilized for the molecular design of new potential antibacterial agents targeting cell wall synthesis.