Intracellular Mycobacterium tuberculosis Exploits Multiple Host Nitrogen Sources during Growth in Human Macrophages.

Nitrogen metabolism of Mycobacterium tuberculosis (Mtb) is crucial for the survival of this important pathogen in its primary human host cell, the macrophage, but little is known about the source(s) and their assimilation within this intracellular niche. Here, we have developed 15N-flux spectral ratio analysis (15N-FSRA) to explore Mtb's nitrogen metabolism; we demonstrate that intracellular Mtb has access to multiple amino acids in the macrophage, including glutamate, glutamine, aspartate, alanine, glycine, and valine; and we identify glutamine as the predominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific amino acid pools, indicating compartmentalized metabolism during intracellular growth. We have discovered that serine is not available to intracellular Mtb, and we show that a serine auxotroph is attenuated in macrophages. This work provides a systems-based tool for exploring the nitrogen metabolism of intracellular pathogens and highlights the enzyme phosphoserine transaminase as an attractive target for the development of novel anti-tuberculosis therapies.

Thymoglobulin Versus Basiliximab Induction Therapy in Low-Risk Kidney Transplant Recipients: A Single-Center Experience.

BACKGROUND: The Kidney Disease: Improving Global Outcomes (KDIGO) guidelines recommend that T-cell-depleting agents should be used only for kidney transplant (KT) recipients at high immunologic risk. However, the effects of thymoglobulin induction therapy in low-immunologic risk KT recipients on tacrolimus, mycophenolic acid, and steroid have not been elucidated yet. METHODS: We retrospectively collected 6 months postoperative clinical data, for low-immunologic risk KT recipients at Soonchunhyang University Hospital. Recipients were divided into thymoglobulin and basiliximab groups, based on the induction agent used. Low-immunologic risk recipients were defined as those with panel-reactive antibody level <30% at the time of kidney transplantation. The incidence of biopsy-proven acute rejection and borderline change was compared between the two groups. RESULTS: Of the 46 low-immunologic risk patients, 25 received thymoglobulin. The incidence of biopsy-proven acute rejection was 0% (n = 0) and that of borderline change was 8% (n = 2) in the thymoglobulin group. The basiliximab group had a significantly higher incidence of rejection (23.8%; n = 5; P = .015) and borderline change (42.9%; n = 9; P = .006). No significant difference in estimated glomerular filtration rate was found between the two groups at 6 months after kidney transplantation. Cytomegalovirus (CMV) infection occurred more frequently in the thymoglobulin group than in the basiliximab group. All patients with CMV infection in both groups were effectively treated with pre-emptive intravenous ganciclovir therapy. CONCLUSIONS: In low-immunologic risk KT recipients who received tacrolimus, mycophenolic acid, and steroid therapy, thymoglobulin induction therapy significantly reduced the incidence of biopsy-proven acute rejection and borderline change compared with basiliximab induction therapy.

Lacrimal gland-derived IL-22 regulates IL-17-mediated ocular mucosal inflammation.

Inflammatory damage of mucosal surface of the eye is a hallmark of dry eye disease (DED) and, in severe cases, can lead to significant discomfort, visual impairment, and blindness. DED is a multifactorial autoimmune disorder with a largely unknown pathogenesis. Using a cross-sectional patient study and a well-characterized murine model of DED, herein we investigated the immunoregulatory function of interleukin-22 (IL-22) in the pathogenesis of DED. We found that IL-22 levels were elevated in lacrimal fluids of DED patients and inversely correlated with severity of disease. Acinar cells of the lacrimal glands (LGs), not inflammatory immune cells, are the primary source of IL-22, which suppresses inflammation in ocular surface epithelial cells upon desiccating stress. Moreover, loss of function analyses using IL-22 knockout mice demonstrated that IL-22 is essential for suppression of ocular surface infiltration of Th17 cells and inhibition of DED induction. Our novel findings elucidate immunoregulatory function of LG-derived IL-22 in inhibiting IL-17-mediated ocular surface epitheliopathy in DED thus making IL-22 a new relevant therapeutic target.

Effects of Gestational Housing on Reproductive Performance and Behavior of Sows with Different Backfat Thickness.

The present study investigated the effects of back-fat thickness at d 107 of gestation and housing types during gestation on reproductive performance and behavior of sows. A total of 64 crossbred sows (Landrace×Yorkshire) in their 3 to 4 parities were allotted to one of four treatments (n = 16) over two consecutive parities. During each parity, sows were assigned to two gestational housing types (stall or group housing) and two level of back-fat thickness (<20 or ≥20) at d 107 of gestation. Gestating sows were transferred from gestational crates to stalls or pens (group housing) 5 weeks before farrowing. All sows were moved to farrowing crates on d 109 of gestation. At weaning, back-fat thickness changes were lesser (p<0.05) in sows having back-fat thickness <20 mm than that of sows with ≥20 mm back-fat thickness at 107 d of gestation. Group housed sows had greater (p<0.05) feed intake and shorter (p<0.05) weaning-to-estrus interval than that of sows in stalls. At weaning, back-fat thickness changes were lesser (p<0.05) in group housed sows than that of sows in stalls. The number of piglets at weaning, growth rate and average daily gain were greater (p<0.05) in group housed sows than that of sows in stalls. During gestation, walking duration was more (p<0.05) in group housed sows. Group housed sows had lesser (p<0.05) farrowing duration and greater (p<0.05) eating time than that of sows in stalls. Result obtained in present study indicated that sows with ≥20 mm back-fat thickness at 107 days had better reproductive performance. Additionally, group housing of sows during last five week of gestation improved the performance and behavior and reproductive efficiency of sows.

Efficiency of professional tooth brushing before ultrasonic scaling.

OBJECTIVES: This study aimed to examine the effect of dental plaque biofilm removal with a toothbrush, an interdental brush and dental floss by a dental hygienist prior to ultrasonic scaling on treatment times and client satisfaction. METHODS: This study was conducted among adults who received scaling after agreeing to participate in this study at a dental clinic in Seoul, Korea, from July to September 2012. Thirty-seven subjects received modified scaling (M-scaling) which is ultrasonic scaling after plaque control with a toothbrush and dental floss by a dental hygienist, and 37 subjects received routine ultrasonic scaling (R-scaling). Univariate and multivariate analyses and chi-squared and t-tests were conducted using SAS. This study was approved by the Kangwon Institutional Review Board. RESULTS: Significant differences were found between the outcomes of M- and R-scaling for both the ultrasonic scaling time (M-scaling, 7.41 ± 6.18 min; R-scaling, 23.22 ± 6.92 min) and the total tooth cleaning time (M-scaling, 15.92 ± 7.70 min; R-scaling, 23.22 ± 6.92 min) (P < 0.001). Subject satisfaction with the scaling process was not significantly different between M-scaling (4.54 ± 0.80) and R-scaling (4.84 ± 0.44). CONCLUSIONS: These findings indicated that removing the dental plaque biofilm with a toothbrush and dental floss by a hygienist before scaling with an ultrasonic device was more effective in reducing the working time of the dental hygienist.

A cross-sectional study to examine the correlation between serum TSH levels and the osteoporosis of the lumbar spine in healthy women with normal thyroid function.

UNLABELLED: We examined the association between serum TSH levels and osteoporosis of the lumbar spine in 756 Korean women aged 65 years or older with normal thyroid function. Low-normal serum TSH levels might be a potential risk factor for the osteoporosis in non-obese elderly women. INTRODUCTION: We aimed to examine the association between serum thyroid-stimulating hormone (TSH) levels and osteoporosis of the lumbar spine in healthy elderly Korean women with normal thyroid function. METHODS: In this cross-sectional study, we evaluated the correlations between serum TSH levels and the osteoporosis of the lumbar spine depending on the body mass index (BMI) in a total of 756 women aged 65 years or older who underwent bone mineral density (BMD) measurement and thyroid function test in a routine health screening examination at our medical institution. RESULTS: After the adjustment of the age and BMI, there was a significant positive correlation between serum TSH level within normal range and the BMD of the lumbar spine (r = 0.165, P < 0.001). In the non-obese elderly women, multivariate-adjusted odds ratios (ORs) for the osteoporosis of the lumbar spine were significantly higher in the 1st and 2nd quartiles of serum TSH levels as compared with their 4th quartile (OR 2.169, 95% CI 1.128-4.171; and OR 2.122, 95% CI 1.123-4.007, respectively). In these women, there were dose-dependent inverse correlations between quartiles of serum TSH levels and the osteoporosis of the lumbar spine (P for trend, 0.008). In the obese women, however, there were no such correlations. CONCLUSIONS: In conclusion, our results suggest that low-normal serum TSH levels might be a potential risk factor for the osteoporosis of the lumbar spine in non-obese elderly women. But further prospective, large-scale, randomized controlled studies are warranted to establish our results.

The economic burden of eating disorders and related mental health comorbidities: An exploratory analysis using the U.S. Medical Expenditures Panel Survey.

BACKGROUND: Very little is known about the economic burden of eating disorders (ED) and related mental health comorbidities. METHODS: Using 5 years of data from the U.S. Medical Expenditures Panel Survey, we estimated the difference in annual health care costs, employment status, and earned income (2011 US$) between individuals with current ED compared to those without ED. We further estimated the contribution of mental health comorbidities to these disparities in health care costs, employment and earnings. RESULTS: Individuals with ED had greater annual health care costs ($1869, p = 0.012), lower but borderline significant employment rates (OR = 0.67, 95% CIs [0.41, 1.09]), and lower but not statistically significant earnings among those who were employed ($2093, p = 0.48), compared to individuals without ED. Among individuals with ED, the presence of mental health comorbidities was associated with higher but not statistically significant health care costs ($1993, p = 0.17), lower borderline significant odds of employment (OR = 0.41, 95% CIs [0.14, 1.20]), and significantly lower earnings ($19,374, p < 0.01). CONCLUSIONS: Treatment and prevention of ED may have broader economic benefits in terms of heath care savings and gains in work productivity than previously recognized. This exploratory study justifies large scale evaluations of the societal economic impact of eating disorders and comorbidities.

The cost-effectiveness of school-based eating disorder screening.

OBJECTIVES: We aimed to assess the value of school-based eating disorder (ED) screening for a hypothetical cohort of US public school students. METHODS: We used a decision-analytic microsimulation model to model the effectiveness (life-years with ED and quality-adjusted life-years [QALYs]), total direct costs, and cost-effectiveness (cost per QALY gained) of screening relative to current practice. RESULTS: The screening strategy cost $2260 (95% confidence interval [CI] = $1892, $2668) per student and resulted in a per capita gain of 0.25 fewer life-years with ED (95% CI = 0.21, 0.30) and 0.04 QALYs (95% CI = 0.03, 0.05) relative to current practice. The base case cost-effectiveness of the intervention was $9041 per life-year with ED avoided (95% CI = $6617, $12,344) and $56,500 per QALY gained (95% CI = $38,805, $71,250). CONCLUSIONS: At willingness-to-pay thresholds of $50,000 and $100,000 per QALY gained, school-based ED screening is 41% and 100% likely to be cost-effective, respectively. The cost-effectiveness of ED screening is comparable to many other accepted pediatric health interventions, including hypertension screening.

Activation of HIF-1α (hypoxia inducible factor-1α) prevents dry eye-induced acinar cell death in the lacrimal gland.

The pathogenesis of immune-mediated lacrimal gland (LG) dysfunction in Sjögren's syndrome has been thoroughly studied. However, the majority of dry eye (DE) is not related to Sjögren type, and its pathophysiology remains unclear. The purpose of this study was to determine and investigate the protective mechanisms against DE stress in mice. DE induced prominent blood vessel loss without apoptosis or necrosis in the LG. Autophagic vacuoles, distressed mitochondria, and stressed endoplasmic reticulum were observed via electron microscopy. Immunoblotting confirmed the increase in autophagic markers. Glycolytic activities were enhanced with increasing levels of succinate and malate that, in turn, activated hypoxia-inducible factor (HIF)-1α. Interestingly, the areas of stable HIF-1α expression overlapped with COX-2 and MMP-9 upregulation in LGs of DE-induced mice. We generated HIF-1α conditional knockout (CKO) mice in which HIF-1α expression was lost in the LG. Surprisingly, normal LG polarities and morphologies were completely lost with DE induction, and tremendous acinar cell apoptosis was observed. Similar to Sjögren's syndrome, CD3(+) and CD11b(+) cells infiltrated HIF-1α CKO LGs. Our results show that DE induced the expression of HIF-1α that activated autophagy signals to prevent further acinar cell damage and to maintain normal LG function.

Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs.

Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva-spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti-G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro-inflammatory cytokines (TNF-α, IL-12 and IL-6). Especially, the truncated rGlBiP containing the heat-shock protein 70 domain-induced cytokine production from mouse DCs. rGlBiP-induced DC activation was initiated by TLR4 in a MyD88-dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF-κB and AP-1. Moreover, CD4(+) T cells stimulated with rGlBiP-treated DCs produced high levels of IL-2 and IFN-γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4-MyD88-p38, ERK1/2 MAPK, NF-κB and AP-1.

Current and potential therapeutic strategies for mucopolysaccharidoses.

WHAT IS KNOWN AND OBJECTIVE: Mucopolysaccharidoses (MPSs) are a group of rare inherited metabolic diseases caused by genetic defects in the production of lysosomal enzymes. MPSs are clinically heterogeneous and are characterized by progressive deterioration in visceral, skeletal and neurological functions. This article aims to review the classification and pathophysiology of MPSs and discuss current therapies and new targeted agents under development. METHODS: A Medline search through PubMed was performed for relevant articles and treatment guidelines on MPSs published in English for years 1970 to September of 2013 inclusive. The references listed in the identified articles, prescribing information of the drugs approved for the treatment of MPSs, as well as recent clinical trial information posted on Clinicaltrials.gov website, were reviewed. RESULTS AND DISCUSSION: Until recently, supportive care was the only option available for the management of MPSs. In the early 2000s, enzyme replacement therapy (ERT) was approved by the United States Food and Drug Administration (FDA) for the treatment of MPS I, II and VI. Clinical trials of ERT showed substantial improvements in patients' somatic symptoms; however, no benefit was found in the neurological symptoms because the enzymes do not readily cross the blood-brain barrier (BBB). Haematopoietic stem cell transplantation (HSCT), another potentially curative treatment, is not routinely advocated in clinical practice due to its high risk profile and lack of evidence for efficacy, except in preserving cognition and prolonging survival in young patients with severe MPS I. In recent years, substrate reduction therapy (SRT) and gene therapy have been rapidly gaining greater recognition as potential therapeutic avenues. WHAT IS NEW AND CONCLUSION: Enzyme replacement therapy (ERT) is effective for the treatment of many somatic symptoms, particularly walking ability and respiratory function, and remains the mainstay of MPS treatment. The usefulness of HSCT has not been established adequately for most MPSs. Although still under investigation, SRT and gene therapy are promising MPS treatments that may prevent the neurodegeneration not affected by ERT.

Age-dependent effects of valproic acid in Alzheimer's disease (AD) mice are associated with nerve growth factor (NGF) regulation.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes cognitive impairment. Major pathophysiological AD characteristics include numerous senile plaque, neurofibrillary tangles, and neuronal loss in the specific regions of patients' brains. In this study, we aimed to understand disease stage-dependent regulation of histone modification for the expression of specific markers in plasma and the hippocampus of in vivo AD model. Since the control of histone acetylation/deacetylation has been studied as one of major epigenetic regulatory mechanisms for specific gene expression, we detected the effects of histone deacetylase (HDAC) inhibitor on marker expression and neuroprotection in in vivo AD model mice. We determined the effects of valproic acid (VPA, HDAC inhibitor), on the levels of cytokines, secreted form of APP (sAPP), nerve growth factor (NGF), and cognitive function in Tg6799 AD mice in three different disease stages (1month: pre-symptomatic; 5months: early symptomatic; and 10months: late-symptomatic stages). VPA decreased the mRNA levels of nuclear factor kappaB (NF-κB) and IL-1ß in the plasma of Tg6799 mice compared to vehicle control at 10months of age. VPA increased the protein levels of NGF in the hippocampus of Tg6799 mice at 5 and 10months of age. In addition, VPA decreased escape latencies of Tg6799 mice at 5 and 10months of age in Morris water maze assessment. Taken together, HDAC inhibition is a promising therapeutic target for AD and it needs to be considered in an age-dependent and/or stage-dependent manner.

Nanometre-scale thermometry in a living cell.

Sensitive probing of temperature variations on nanometre scales is an outstanding challenge in many areas of modern science and technology. In particular, a thermometer capable of subdegree temperature resolution over a large range of temperatures as well as integration within a living system could provide a powerful new tool in many areas of biological, physical and chemical research. Possibilities range from the temperature-induced control of gene expression and tumour metabolism to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biological processes at the subcellular level. Here we demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin associated with nitrogen-vacancy colour centres in diamond. Our technique makes it possible to detect temperature variations as small as 1.8 mK (a sensitivity of 9 mK Hz(-1/2)) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centres in diamond nanocrystals (nanodiamonds), we directly measure the local thermal environment on length scales as short as 200 nanometres. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, we demonstrate temperature-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences.

Risk factors for overgrowth after flexible intramedullary nailing for fractures of the femoral shaft in children.

We analysed retrospectively the risk factors leading to femoral overgrowth after flexible intramedullary nailing in 43 children (mean age 7.1 years (3.6 to 12.0)) with fractures of the shaft of the femur. We reviewed their demographic data, mechanism of injury, associated injuries, the type and location of the fractures, the nail-canal diameter (NCD) ratios and femoral overgrowth at a mean follow-up of 40.7 months (25.2 to 92.7). At that time, the children were divided into two groups, those with femoral overgrowth of < 1 cm (Group 1), and those with overgrowth of ≥ 1 cm (Group 2). The mean femoral overgrowth of all patients was 0.6 cm at final follow-up. Overgrowth of ≥ 1 cm was noted in 11 children (25.6%). The NCD ratio was significantly lower in Group 2 than in Group 1, with an odds ratio of 30.0 (p = 0.003). We believe that a low NCD ratio is an indicator of an unstable configuration with flexible intramedullary nailing, and have identified an association between a low NCD ratio and femoral overgrowth resulting in leg-length discrepancy after flexible intramedullary nailing in paediatric femoral shaft fractures.

EphA/ephrin-A signaling is critically involved in region-specific apoptosis during early brain development.

EphAs and ephrin-As have been implicated in the morphogenesis of the developing brain. We found that EphA7 and ephrin-A5 are coexpressed in the dorsal midline (DM) of the diencephalon and anterior mesencephalon. Interestingly, programmed cell death (PCD) of the neural epithelial cells normally found in this region was reduced in ephrin-A5/ephrin-A2 dual-deficient embryos. In contrast, in vivo expression of ephrin-A5-Fc or full-length ephrin-A5 strongly induced apoptosis in neural epithelial cells and was accompanied by severe brain malformation during embryonic development. Expression of ephrinA5-Fc correlated with apoptosis of EphA7-expressing cells, whereas null mutation of ephrin-A5 resulted in the converse phenotype. Importantly, null mutation of caspase-3 or endogenous ephrin-A5 attenuated the PCD induced by ectopically overexpressed ephrin-A5. Together, our results suggest that brain region-specific PCD may occur in a region where EphAs cluster with neighboring ephrin-As through cell-cell contact.

Interleukin enhancer-binding factor 3 promotes breast tumor progression by regulating sustained urokinase-type plasminogen activator expression.

Sustained urokinase-type plasminogen activator (uPA) expression is detected in aggressive breast tumors. Although uPA can be transiently upregulated by diverse extracellular stimuli, sustained, but not transiently upregulated uPA expression contributes to breast cancer invasion/metastasis. Unfortunately, how sustained uPA expression is achieved in invasive/metastatic breast cancer cells is unknown. Here, we show that sustained and transiently upregulated uPA expression are regulated by distinct mechanisms. Using a collection of transcription factor-targeted small-interfering RNAs, we discovered that interleukin enhancer-binding factor 3 (ILF3) is required for sustained uPA expression. Two discrete mechanisms mediate ILF3 action. The first is that ILF3 activates uPA transcription by binding to the CTGTT sequence in the nucleotides -1004∼-1000 of the uPA promoter; the second is that ILF3 inhibits the processing of uPA mRNA-targeting primary microRNAs (pri-miRNAs). Knockdown of ILF3 led to significant reduction in in vitro cell growth/migration/invasion and in vivo breast tumor development. Importantly, immunohistochemistry (IHC) showed that nuclear ILF3, but not cytoplasmic ILF3 staining correlates with elevated uPA level and higher grades of human breast tumor specimens. Nuclear localization of ILF3 highlights the role of ILF3 in sustained uPA expression as a transcription activator and pri-miRNA processing blocker. In conclusion, this study shows that ILF3 promotes breast tumorigenicity by regulating sustained uPA expression.

Vascularisation of myringo-/tympanoplastic grafts in active and inactive chronic mucosal otitis media: a prospective cohort study.

OBJECTIVES: To evaluate the vascularisation process of the grafted fascia or perichondrium in active and inactive chronic mucosal otitis media. DESIGN: Prospective study. SETTING: University-based, secondary referral hospital. PARTICIPANTS: Two hundred thirty-two patients who underwent type 1 tympanoplasty or myringoplasty by one experienced surgeon. MAIN OUTCOME MEASURES: After postoperative days 5-7, the graft was inspected using a binocular operating microscope at least twice weekly until vascularisation was confirmed to have commenced. This point was defined as the time of vascularisation. An intact graft at 3 months postoperatively was considered a closure success. RESULTS: The mean time of vascularisation of 232 grafts was 14.2 ± 3.6 days (range 8-25). The vascularisation time differed according to the perforation size. The perforations involving 50% and less of the pars tensa were revascularised earlier than those involving 75% and more. There was no relationship between vascularisation time and closure success or failure. Age itself did not influence the vascularisation time or the risk of closure failure. Postoperative otorrhea was higher in patients with a preoperative wet middle ear mucosa than among those with a dry one. CONCLUSION: The vascularisation time was shorter in perforations of 50% and less than those of 75% and more but the rate of closure success was not different between two. The graft failure was not attributed to the failure of vascularisation. Age itself seemed not to be a contraindication for myringo-/tympanoplasty in otherwise healthy elderly patients.

Short-range order and near-field effects on optical scattering and structural coloration.

We have investigated wavelength-dependent light scattering in biomimetic structures with short-range order. Coherent backscattering experiments are performed to measure the transport mean free path over a wide wavelength range. Overall scattering strength is reduced significantly due to short-range order and near-field effects. Our analysis explains why single scattering of light is dominant over multiple scattering in similar biological structures and is responsible for color generation.