bluuebunny/crossref_metadata_2025 huggingface dataset:
Risk scores for diabetes and impaired glycaemia in the Middle East and North Africa
Authors: Handlos L. N. , Witte D. R. , Almdal T. P. , Nielsen L. B. , Badawi S. E. , Sheikh A. R. A. , Belhadj M. , Nadir D. , Zinai S. , Vistisen D.
Published: January 2013

Aims

To develop risk scores for diabetes and diabetes or impaired glycaemia for individuals living in the Middle East and North Africa region. In addition, to derive national risk scores for Algeria, Saudi Arabia and the United Arab Emirates and to compare the performance of the regional risk scores with the national risk scores.

Methods

An opportunistic sample of 6588 individuals aged 30–75 years was screened. Screening consisted of a questionnaire and a clinical examination including measurement of HbA1c. Two regional risk scores and national risk scores for each of the three countries were derived separately by stepwise backwards multiple logistic regression with diabetes [HbA1c ≥ 48 mmol/mol (≥ 6.5%)] and diabetes or impaired glycaemia [HbA1c ≥ 42 mmol/mol (≥ 6.0%)] as outcome. The performance of the regional and national risk scores was compared in data from each country by receiver operating characteristic analysis.

Results

The eight risk scores all included age and BMI, while additional variables differed between the scores. The areas under the receiver operating characteristic curves were between 0.67 and 0.70, and for sensitivities approximately 75%; specificities varied between 50% and 57%. The regional and the national risk scores performed equally well in the three national samples.

Conclusions

Two regional risk scores for diabetes and diabetes or impaired glycaemia applicable to the Middle East and North Africa region were identified. The regional risk scores performed as well as the national risk scores derived in the same manner.
Prevalence of diabetic retinopathy in Type 2 diabetes in developing and developed countries
Authors: Ruta L. M. , Magliano D. J. , LeMesurier R. , Taylor H. R. , Zimmet P. Z. , Shaw J. E.
Published: January 2013

Background

As the global prevalence of diabetes increases, so will the numbers of people with diabetic retinopathy. Our review aimed to provide a comprehensive picture of available studies of diabetic retinopathy and how prevalence varies around the developed and developing world.

Methods

A detailed literature search using PubMed was undertaken. The following search term was used: ‘diabetic retinopathy AND prevalence’. The titles and abstracts of all publications identified by the search were reviewed and 492 studies were retrieved. Inclusion and exclusion criteria were applied.

Results

A total of 72 articles from 33 countries were included. There were only 26 population‐based studies using fundus photography (12 in developing countries), of which only 16 (eight in developing countries) were published since 2000. Prevalence estimates varied from as low as 10% to as high as 61% in persons with known diabetes and from 1.5 to 31% in newly diagnosed diabetes. Across all the studies, the median (interquartile range) prevalence of any diabetic retinopathy in known diabetes was 27.9% (22–37%) and 10.5% (6–16%) in newly diagnosed diabetes. Prevalence of diabetic retinopathy was higher in developing countries.

Conclusion

Significant gaps exist in that reliable population‐based data from developing nations and indigenous populations in particular are lacking. Major differences in study characteristics and methodologies make comparisons very difficult. More research is required and study methodologies must be better standardized. This will provide important information for prevention and treatment strategies.
Lack of GPR40/FFAR1 does not induce diabetes even under insulin resistance condition
Authors: Matsuda‐Nagasumi K. , Takami‐Esaki R. , Iwachidow K. , Yasuhara Y. , Tanaka H. , Ogi K. , Nakata M. , Yano T. , Hinuma S. , Taketomi S. , Odaka H. , Kaisho Y.
Published: January 2013

Aims

G protein‐coupled receptor/free fatty acid receptor 1 (GPR40/FFAR1) regulates free fatty acid‐induced insulin secretion. This study has been performed to clarify whether or not loss of GPR40/FFAR1 function exacerbates diabetes, that is, whether GPR40 has an essential physiological role in the development of diabetes or not.

Methods

We generated GPR40/FFAR1 knockout (KO) mice and analysed their phenotypes in vitro and in vivo under the condition of dietary or genetically induced insulin resistance.

Results

GPR40/FFAR1 KO mice kept on a high‐fat diet became obese, developed glucose intolerance to a similar degree as GPR40/FFAR1 wild‐type (WT) mice. In addition, the phenotype of KO mice harbouring diabetogenic KK background genes showed glucose intolerance at a level similar to level for control KK mice. In both mouse models with insulin resistance, insulin secretion after oral glucose load and homeostasis model assessment‐insulin resistance (HOMA‐IR) did not change between GPR40/FFAR1 KO and WT mice. Although glucose‐induced insulin secretion under high palmitate concentration was significantly lower in KO than in WT islets, pancreatic insulin content and insulin secretion stimulated with glucose alone were not different between KO and WT mice.

Conclusions

GPR40/FFAR1 has a major role in regulating fatty‐acid‐mediated insulin secretion, but the lack of GPR40/FFAR1 does not exacerbate glucose intolerance and insulin resistance induced by high‐fat diet or diabetogenic KK gene. Our findings indicate that loss of GPR40/FFAR1 function does not play an important role in inducing or exacerbating diabetes.
Interleukin‐17 enhances the production of interferon‐γ and tumour necrosis factor‐α by bone marrow T lymphocytes from patients with lower risk myelodysplastic syndromes
Authors: Zhang Zheng , Li Xiao , Guo Juan , Xu Feng , He Qi , Zhao Youshan , Yang Yujuan , Gu Shucheng , Zhang Yan , Wu Lingyun , Chang Chunkang
Published: January 2013

Introduction

Lower risk myelodysplastic syndromes (MDSs) are characterised by increased apoptosis of haematopoietic cells in the bone marrow (BM). The mechanism driving this excessive apoptosis involves multiple immune molecules, including inflammatory cytokines such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukins (ILs). Interleukin‐17 (IL‐17) is the hallmark cytokine produced by CD4+ Th17 cells, and IL‐17 mediates activation of the adaptive T‐cell response inducing an inflammatory cytokine environment. However, little is known about the role of IL‐17 in MDS‐associated immune dysfunction.

Methods

A total of 47 patients with myelodysplastic syndromes were enrolled in this study, and the levels of IL‐17 and IL‐17 receptor (IL‐17R) in BM mononuclear cells (BMNCs) were detected by real‐time polymerase chain reaction (RQ‐PCR) and enzyme‐linked immunosorbent assay (ELISA). Then, BMNCs were stimulated with recombinant human IL‐17 (rhIL‐17), and flow cytometry was used to analyse the production of IFN‐γ and TNF‐α by CD4+ and CD+ T lymphocytes from patients with lower‐risk MDS. Characterisation of IL‐17 expression in patients with the HLA‐DR15 allele or hypocellularity was also performed.

Results

mRNA levels for both IL‐17 and the IL‐17R subunits in BMNCs and for IL‐17 in the BM and plasma were higher in patients with lower‐risk MDS as compared to patients with higher‐risk MDS and normal controls. The production of IFN‐γ and TNF‐α by CD4+ and CD8+ T lymphocytes from patients with lower‐risk MDS could be enhanced by recombinant human IL‐17 (rhIL‐17) treatment. Furthermore, increased IL‐17 expression was associated with more severe anaemia in with patients with MDS.

Conclusion

Elevated IL‐17 levels and IL‐17‐induced IFN‐γ and TNF‐α overproduction may be involved in the pathogenesis of lower risk MDS.
Prognostic significance of programmed cell death‐1‐positive cells in follicular lymphoma patients may alter in the rituximab era
Authors: Takahashi Hiroyuki , Tomita Naoto , Sakata Seiji , Tsuyama Naoko , Hashimoto Chizuko , Ohshima Rika , Matsuura Shiro , Ogawa Koji , Yamamoto Wataru , Kameda Yoichi , Enaka Makiko , Inayama Yoshiaki , Kasahara Masao , Takekawa Yoshinori , Onoda Noboru , Motomura Shigeki , Ishigatsubo Yoshiaki , Takeuchi Kengo
Published: January 2013

Programmed cell death‐1 (PD‐1) is involved in one of the inhibitory pathways of the B7‐cluster of differentiation (CD) 28 family; this pathway is known to be involved in the attenuation of T‐cell responses and promotion of T‐cell tolerance. PD‐1 is known to negatively regulate T‐cell receptor‐mediated proliferation and cytokine production, lead to alternation in the tumor microenvironment. Although several studies have shown that high levels of PD‐1‐positive cells in follicular lymphoma (FL) patients influence their prognosis, those studies included patients treated without rituximab, and the prognostic impact of PD‐1 positivity in the rituximab era (R‐era) has not yet been elucidated. We retrospectively studied 82 patients with FL uniformly treated with standard R‐CHOP therapy at six institutions between 2001 and 2009 (median follow‐up for survivors: 55 months). We also collected and examined biopsy specimens for diagnosis with respect to PD‐1 positivity. The PD‐1 positivity was significantly higher in male patients and patients with high beta‐2 microglobulin (B2M ≥ 3.0) (P = 0.03 and 0.003, respectively). Three‐year progression free survival (PFS) and overall survival (OS) were 60% and 86%, respectively. By univariate analysis, elevated LDH (P = 0.07) worsened PFS. Male gender (P = 0.03), high FLIPI score (P = 0.05), and high B2M levels (P = 0.08) worsened OS. Multivariate analysis detected no significant prognostic factors, including PD‐1 positivity. However, in male subgroup, high levels of PD‐1‐positive cells were found to be a prognostic factor for PFS. Addition of rituximab might have altered the prognostic impact of PD‐1‐positive cells.
Thirty‐year experience in preventing haemoglobinopathies in Greece: achievements and potentials for optimisation
Authors: Ladis Vassilis , Karagiorga‐Lagana Markissia , Tsatra Ioanna , Chouliaras Giorgos
Published: January 2013

Objectives

Beta thalassaemia major (β‐TM) and sickle‐cell disease (SCD) are severe haemogobinopathies requiring life‐lasting, advanced medical management. In the Mediterranean region, both conditions occur with high frequency. We assessed the efficacy of the National Program for the Prevention of Haemoglobinopathies in Greece during the last 30 yrs.

Methods

Data of affected births between 01/01/1980 and 31/12/2009 were collected in a nationwide scale, and expected vs. observed rates of new births were calculated and compared. In a subpopulation of affected births of Greek origin, the causes for occurrence of the new affected birth were also collected and analysed.

Results

Overall, the reduction in new cases was 81.1% and 84.6% for β‐TM and SCD, respectively. For β‐TM, a constant declining trend was recorded over the 30‐yr period, whereas for SCD, a transient reversal was observed in the mid‐1990s probably due to the significant influx of immigrants of African origin. Programme failure was 2.2 times more common among new β‐TM births of Greek origin compared to new SCD cases (P < 0.001). Unawareness and parental choice were more frequent in SCD compared to β‐TM (unawareness: OR = 1.4, P = 0.05, parental choice: OR = 1.9, P = 0.01). The main cause for programme failure was carrier misidentification and incorrect genetic advice for β‐TM and SCD, respectively.

Conclusions

The β‐TM and SCD prevention programme in Greece has significantly reduced the numbers of new affected births. The outcomes could be optimised in groups of non‐Greek origin, in carrier identification and by offering specialised genetic counselling.
Attention deficit hyperactivity disorder and dental anxiety in adults: relationship with oral health
Authors: Carlsson Viktor , Hakeberg Magnus , Blomkvist Klas , Wide Boman Ulla
Published: January 2013

The aim of the present study was to investigate the presence of attention deficit hyperactivity disorder (ADHD) in adult patients with severe dental anxiety. Specifically, we analysed the relationship among ADHD, oral health, and dental anxiety. The World Health Organization Adult ADHD Self‐Report Scale (ASRS) Screener was administered to a consecutive sample of patients referred to a dental fear research and treatment clinic. Patients completed questionnaires measuring dental anxiety (Dental Fear Survey) and self‐rated oral health, and underwent a full radiographic examination. Of the total sample (n = 110), 16% scored above the established ASRS cut‐off point, which is indicative of having ADHD. The ADHD group showed a higher level of dental anxiety and poorer self‐rated oral health. There were also indications of poorer clinical oral health in the ADHD group, but these results did not reach statistical significance. In conclusion, the results of this study indicate an increased prevalence of ADHD in highly dentally anxious adults and the need to pay special attention to these patients because of greater treatment needs and increased dental anxiety.
Dentists' skills with fearful patients: education and treatment
Authors: Brahm Carl‐Otto , Lundgren Jesper , Carlsson Sven G. , Nilsson Peter , Hultqvist Johanna , Hägglin Catharina
Published: January 2013

The aims were to explore dentists' skills in dental fear, current strategies when treating fearful adult patients, and the possible need for additional education among dentists working in Sweden. A sample of 1,293 members of the Association of Public Health Dentists in Sweden were asked to respond to a Web survey concerning dental fear. The response rate was 69% (n = 889); 91% trained in Sweden and 9% trained in another country. The most frequently used pharmacological anxiety‐reducing techniques were medication with a midazolame mixture (72%) and benzodiazepine tablets (77%), and the most commonly used psychological techniques were relaxation (68%), distraction (66%), and Tell‐Show‐Do (86%). A larger proportion of dentists trained in Sweden, compared with dentists who were trained in other countries, reported that they had received undergraduate training in dental fear. Dentists trained in Sweden more often reported competence in pharmacological and psychological anxiety‐reducing techniques, compared with dentists who were trained in other countries. Higher levels of self‐rated efficacy in treating fearful patients accompanied additional education in dental fear after graduation. In conclusion, Swedish dentists use a variety of techniques to meet the needs of fearful dental patients. Competence in anxiety‐reducing techniques is associated with self‐efficacy and the site of education.
A lentiviral vector‐based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin
Authors: Staunstrup Nicklas Heine , Bak Rasmus O. , Cai Yujia , Svensson Lars , Petersen Thomas K. , Rosada Cecilia , Stenderup Karin , Bolund Lars , Mikkelsen Jacob Giehm
Published: January 2013

Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus‐directed gene delivery to xenotransplanted human skin with vitamin D3‐induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA‐binding domain, the vitamin D3‐responsive sensor facilitates non‐invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto ‘sensorized’ human skin, the sensor produces a drug‐induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non‐invasive in vivo screening system for further development and refinement of vitamin D3 analogues.
Aggravation of atopic dermatitis‐like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice
Authors: Kim Ji‐Yun , Jeong Mi Sook , Park Kui Young , Seo Seong Jun
Published: January 2013

Formaldehyde (FA) has been known to be associated with development of asthma (AS) and atopic dermatitis (AD). In this study, we investigated whether FA inhalation would affect the provocation or exacerbation of AD‐like symptoms. Atopic‐prone NC/Nga mice were exposed to low (0.2 ppm) and high (1.0 ppm) concentration of FA by inhalation. Combined exposure to low concentration of FA inhalation and topical house dust mite (HDM) stimulation significantly upregulated HDM‐induced total plasma IgE and IgG2a production, Th1‐, Th2‐, Th17‐related cytokine as well as COX‐2 mRNA expressions in the skin. Interestingly, independent FA inhalation, especially at low concentration (0.2 ppm), increased the skin mRNA expressions of IL‐13, IL‐17E/IL‐25 and COX‐2, even though it failed to induce AD‐like skin inflammation. In conclusion, we suggest that increased skin mRNA expressions of IL‐13, IL‐25/IL‐17E and COX‐2 by independent low concentration of FA exposure might be a key factor to exacerbate HDM‐mediated AD‐like skin inflammation.
Cutaneous lymphocyte‐associated antigen as a novel predictive marker of TNF‐alpha inhibitor biological therapy in psoriasis
Authors: Jókai Hajnalka , Szakonyi József , Kontár Orsolya , Barna Gábor , Inotai Dóra , Kárpáti Sarolta , Holló Péter
Published: January 2013

A considerable number of patients with psoriasis show secondary resistance during long‐term TNF‐alpha inhibitor therapy, necessitating the identification of reliable predictive markers. Predictive role of cutaneous lymphocyte‐associated antigen (CLA) was investigated. Thirty‐eight severe patients with psoriasis were treated for a 24‐week‐long study period. Clinical responsiveness (PASI) and changes in flow cytometry–measured peripheral lymphocyte CLA expression (week 0–2–6) were statistically analysed. Regarding 24‐week‐long treatment outcome patients were divided into two groups: During the first 6 weeks, mean CLA expression showed significant (P = 0.034604) increase among responders (32/38), while after a preliminary increase, it was significantly (P = 0.012539) decreasing in the relapsing group (6/38). Pearson's correlation analysis showed significant negative correlation between PASI and CLA changes. Responders showed (not significantly) lower initial CLA expression than relapsing patients. Our observations suggest change in CLA expression during the first 6 weeks of induction period to serve as a potential predictive marker of TNF‐alpha inhibitor therapy in psoriasis.
P2X7 receptor is required for neutrophil accumulation in a mouse model of irritant contact dermatitis
Authors: da Silva Gabriela L. , Sperotto Nathalia D. M. , Borges Thiago J. , Bonorino Cristina , Takyia Cristina M. , Coutinho‐Silva Robson , Campos Maria M. , Zanin Rafael F. , Morrone Fernanda B.
Published: January 2013

Irritant contact dermatitis (ICD) is an inflammatory reaction caused by chemical toxicity on the skin. The P2X7 receptor (P2X7R) is a key mediator of cytokine release, which recruits immune cells to sites of inflammation. We investigated the role of P2X7R in croton oil (CrO)‐induced ICD using in vitro and in vivo approaches. ICD was induced in vivo by CrO application on the mouse ear and in vitro by incubation of murine macrophages and dendritic cells (DCs) with CrO and ATP. Infiltrating cells were identified by flow cytometry, histology and myeloperoxidase (MPO) determination. Effects of the ATP scavenger apyrase were assessed to investigate further the role of P2X7R in ICD. Animals were also treated with N‐1330, a caspase‐1 inhibitor, or with clodronate, which induces macrophage apoptosis. CrO application induced severe inflammatory Gr1+ cell infiltration and increased MPO levels in the mouse ear. Selective P2X7R antagonism with A438079 or genetic P2X7R deletion reduced the neutrophil infiltration. Clodronate administration significantly reduced Gr1+ cell infiltration and local IL‐1β levels. In vitro experiments confirmed that A438079 or apyrase treatment prevented the increase in IL‐1β that was evoked by macrophage and DC incubation with CrO and ATP. These data support a key role for P2X7 in ICD‐mediated inflammation via modulation of inflammatory cells. It is tempting to suggest that P2X7R inhibition might be an alternative ICD treatment.
Vascular endothelial‐cadherin stimulates syndecan‐1‐coupled insulin‐like growth factor‐1 receptor and cross‐talk between αVβ3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis
Authors: Rapraeger Alan C. , Ell Brian J. , Roy Madhuchhanda , Li Xuehui , Morrison Orrianne R. , Thomas Grant M. , Beauvais DeannaLee M.
Published: January 2013

Vascular endothelial growth factor (VEGF)‐stimulated angiogenesis depends on a cross‐talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)‐cadherin and the αVβ3 integrin. Because we have shown that αVβ3 integrin activation is dependent on its incorporation, along with the insulin‐like growth factor‐1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan‐1 (Sdc1), we questioned the role of this core complex in VEGF‐stimulated angiogenesis. We find that the Sdc1‐coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE‐cadherin) engagement. VE‐cadherin binding to Fc/VE‐cadherin extracellular domain chimera activates Sdc1‐coupled IGF1R and αvβ3 integrin; this depends on VEGFR2 and c‐Src activated by the cadherin. Blocking homotypic VE‐cadherin engagement disrupts VEGF‐stimulated cell migration, which is restored by clustering the cadherin in the absence of cell–cell adhesion. This cadherin‐dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)92–119, a peptide that competitively disrupts the Sdc1‐coupled ternary complex and prevents the αVβ3 integrin activation required for VEGFR2 activation. VEGFR2‐stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVβ3 integrin comprises a core activation mechanism activated by VE‐cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.
AKT signalling is required for ribosomal RNA synthesis and progression of Eμ‐Myc B‐cell lymphoma in vivo
Authors: Devlin Jennifer R. , Hannan Katherine M. , Ng Pui Y. , Bywater Megan J. , Shortt Jake , Cullinane Carleen , McArthur Grant A. , Johnstone Ricky W. , Hannan Ross D. , Pearson Richard B.
Published: January 2013

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a ‘super signalling’ network mediating malignancy. A fundamental downstream action of this signalling network is up‐regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC‐driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC‐driven model of B‐cell lymphoma (Eμ‐Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK‐2206 rapidly and potently antagonizes rDNA transcription in Eμ‐Myc B‐cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour‐bearing mice with MK‐2206 resulted in a significant delay in disease progression, associated with increased B‐cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.
The regulatory roles of small leucine‐rich proteoglycans in extracellular matrix assembly
Authors: Chen Shoujun , Birk David E.
Published: January 2013

Small leucine‐rich proteoglycans (SLRPs) are involved in a variety of biological and pathological processes. This review focuses on their regulatory roles in matrix assembly. SLRPs have protein cores and hypervariable glycosylation with multivalent binding abilities. During development, differential interactions of SLRPs with other molecules result in tissue‐specific spatial and temporal distributions. The changing expression patterns play a critical role in the regulation of tissue‐specific matrix assembly and therefore tissue function. SLRPs play significant structural roles within extracellular matrices. In addition, they play regulatory roles in collagen fibril growth, fibril organization and extracellular matrix assembly. Moreover, they are involved in mediating cell–matrix interactions. Abnormal SLRP expression and/or structures result in dysfunctional extracellular matrices and pathophysiology. Altered expression of SLRPs has been found in many disease models, and structural deficiency also causes altered matrix assembly. SLRPs regulate assembly of the extracellular matrix, which defines the microenvironment, modulating both the extracellular matrix and cellular functions, with an impact on tissue function.
Use of ‘small but smart’ libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran‐3‐yl acetate
Authors: Nobili Alberto , Gall Markus G. , Pavlidis Ioannis V. , Thompson Mark L. , Schmidt Marlen , Bornscheuer Uwe T.
Published: January 2013

Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran‐3‐yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a ‘small but smart’ concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of α/β‐hydrolase fold enzymes with the bioinformatic tool 3dm. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site‐saturation mutagenesis approach. Whereas the wild‐type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)‐tetrahydrofuran‐3‐yl acetate, the best variants obtained with this approach showed increased E‐values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.
Evidence that Na+/H+ exchanger 1 is an ATP‐binding protein
Authors: Shimada‐Shimizu Naoko , Hisamitsu Takashi , Nakamura Tomoe Y. , Wakabayashi Shigeo
Published: January 2013

Na+/H+ exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP‐binding protein. We designed a baculovirus vector carrying both epitope‐tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1–CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8‐azido‐ATP‐biotin. UV irradiation promoted the incorporation of 8‐azido‐ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µm in the presence of Mg2+. The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mm, close to the ATP concentration giving the half‐maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8‐azido‐ATP‐binding site is within the C‐terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1‐derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542–Pro598), which is critical for ATP‐dependent regulation of NHE1. These findings suggest that NHE1 is an ATP‐binding transporter. Thus, ATP may serve as a direct activator of NHE1.
The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual‐step process and contains proteins involved in plant defence
Authors: Scarafoni Alessio , Ronchi Alessandro , Prinsi Bhakti , Espen Luca , Assante Gemma , Venturini Giovanni , Duranti Marcello
Published: January 2013

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre‐formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post‐extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual‐step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.
Aromatic substitution of the FAD‐shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase
Authors: Meints Carla E. , Simtchouk Svetlana , Wolthers Kirsten R.
Published: January 2013

Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and kcat/Km(NADPH) for steady‐state cytochrome c3+ reduction and a 10‐fold increase in the rate constant (kobs1) associated with hydride transfer. Elevated primary kinetic isotope effects on kobs1 for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c3+ reduction, a 30‐ and 3.5‐fold decrease in the rate of FAD reduction, accumulation of a FADH2–NADP+ charge‐transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is ‘gated’ by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.
Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies
Authors: Terrill Jessica R. , Radley‐Crabb Hannah G. , Iwasaki Tomohito , Lemckert Frances A. , Arthur Peter G. , Grounds Miranda D.
Published: January 2013

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin‐deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin‐deficient mdx mice.
Recombinant purine nucleoside phosphorylases from thermophiles: preparation, properties and activity towards purine and pyrimidine nucleosides
Authors: Zhou Xinrui , Szeker Kathleen , Janocha Bernd , Böhme Thomas , Albrecht Dirk , Mikhailopulo Igor A. , Neubauer Peter
Published: January 2013

Thermostable nucleoside phosphorylases are attractive biocatalysts for the synthesis of modified nucleosides. Hence we report on the recombinant expression of three ‘high molecular mass’ purine nucleoside phosphorylases (PNPs) derived from the thermophilic bacteria Deinococcus geothermalis, Geobacillus thermoglucosidasius and from the hyperthermophilic archaeon Aeropyrum pernix (5′–methythioadenosine phosphorylase; ApMTAP). Thermostability studies, kinetic analysis and substrate specificities are reported. The PNPs were stable at their optimal temperatures (DgPNP, 55 °C; GtPNP, 70 °C; ApMTAP, activity rising to 99 °C). Substrate properties were investigated for natural purine nucleosides [adenosine, inosine and their C2′‐deoxy counterparts (activity within 50–500 U·mg−1)], analogues with 2′‐amino modified 2′‐deoxy‐adenosine and ‐inosine (within 0.1–3 U·mg−1) as well as 2′‐deoxy‐2′‐fluoroadenosine (9) and its C2′‐arabino diastereomer (10, within 0.01–0.03 U·mg−1). Our results reveal that the structure of the heterocyclic base (e.g. adenine or hypoxanthine) can play a critical role in the phosphorolysis reaction. The implications of this finding may be helpful for reaction mechanism studies or optimization of reaction conditions. Unexpectedly, the diastereomeric 2′‐deoxyfluoro adenine ribo‐ and arabino‐nucleosides displayed similar substrate properties. Moreover, cytidine and 2′‐deoxycytidine were found to be moderate substrates of the prepared PNPs, with substrate activities in a range similar to those determined for 2′‐deoxyfluoro adenine nucleosides 9 and 10. C2′‐modified nucleosides are accepted as substrates by all recombinant enzymes studied, making these enzymes promising biocatalysts for the synthesis of modified nucleosides. Indeed, the prepared PNPs performed well in preliminary transglycosylation reactions resulting in the synthesis of 2′‐deoxyfluoro adenine ribo‐ and arabino‐ nucleosides in moderate yield (24%).
Inflammatory vulnerability associated with the rh5‐HTTLPR genotype in juvenile rhesus monkeys
Authors: Amaral W. Z. , Lubach G. R. , Bennett A. J. , Coe C. L.
Published: January 2013

Individual variation in serotonergic function is associated with reactivity, risk for affective disorders, as well as an altered response to disease. Our study used a nonhuman primate model to further investigate whether a functional polymorphism in the promoter region for the serotonin transporter gene helps to explain differences in proinflammatory responses. Homology between the human and rhesus monkey polymorphisms provided the opportunity to determine how this genetic variation influences the relationship between a psychosocial stressor and immune responsiveness. Leukocyte numbers in blood and interleukin‐6 (IL‐6) responses are sensitive to stressful challenges and are indicative of immune status. The neutrophil‐to‐lymphocyte ratio and cellular IL‐6 responses to in vitro lipopolysaccharide stimulation were assessed in 27 juvenile male rhesus monkeys while housed in stable social groups (NLL = 16, NS = 11) and also in 18 animals after relocation to novel housing (NLL = 13, NS = 5). Short allele monkeys had significantly higher neutrophil‐to‐lymphocyte ratios than homozygous Long allele carriers at baseline [t(25) = 2.18, P = 0.02], indicative of an aroused state even in the absence of disturbance. In addition, following the housing manipulation, IL‐6 responses were more inhibited in short allele carriers (F1,16 = 8.59, P = 0.01). The findings confirm that the serotonin transporter gene‐linked polymorphism is a distinctive marker of reactivity and inflammatory bias, perhaps in a more consistent manner in monkeys than found in many human studies.
Oral health of dental assistants and patients receiving maintenance – an investigation based on a district of Thuringia, Germany
Authors: Ziebolz D , Klopfleisch S , Fresmann S , Hornecker E , Mausberg RF
Published: January 2013

Objectives

The aim of the study was to assess data for the oral health behaviour and oral health of dental assistants (DAs) and patients (PTs) who regularly received preventive dental care.

Methods

One‐hundred DAs (38.8 years) and 100 PTs (44 years) participated in the study conducted in the Unstrut‐Hainich region of Thuringia, Germany. A questionnaire established oral hygiene and smoking habits. The dental examination included the caries index DMF‐T, evaluation of the periodontal situation (PSI) as well as gingival inflammation [papilla bleeding index (PBI)].

Results

Seventy‐seven DAs and 46 PTs used dental floss regularly. Twenty‐four DAs and 27 PTs were smokers. The mean DMF‐T of DAs was 12.5, which was significantly lower than the DMF‐T of PTs (17.0) (P < 0.001). The main reason for this difference was the number of missing teeth (DAs: 0.9, PTs: 4.8; P < 0.001). In both groups, several participants required periodontal treatment. Nevertheless, the number of DAs with PSI scores 3 or 4 was significantly lower (9%) than the corresponding number of PTs (63%) (P < 0.001). The PBI showed a significant difference between the groups (DAs = 0.1, PTs = 0.3; P < 0.01).

Conclusions

Although the oral health of the participating DAs clearly exceeded the results found for PTs, it was concluded that DAs did not avail themselves of their job‐related knowledge and skills and so failed to optimize their personal oral health behaviour.
Prevalence of and risk factors for isolation of meticillin‐resistant Staphylococcus spp. from dogs with pyoderma in northern California, USA
Authors: Eckholm Nicole G. , Outerbridge Catherine A. , White Stephen D. , Sykes Jane E.
Published: January 2013

Background –  Canine pyodermas associated with meticillin‐resistant Staphylococcus spp. (MRS) have increased in prevalence over the past decade.

Hypothesis/Objectives –  To compare the prevalence of MRS isolation from dogs with superficial pyoderma at a primary care clinic (PCC) and those at a tertiary care facility (VMTH) in California, USA, and identify associated risk factors.

Animals –  Client‐owned dogs from the VMTH (80 dogs) and the PCC (30 dogs).

Methods –  Aerobic bacterial culture and antibiotic susceptibility were performed on swab specimens collected from dogs, and meticillin resistance was determined using microdilution methods according to Clinical and Laboratory Standards Institute guidelines. A mecA gene PCR assay was used to confirm meticillin resistance when possible.

Results –  Of 89 staphylococcal isolates from the VMTH, 34 (38.2%) were meticillin resistant. In 31 dogs, pyoderma persisted, and one or more follow‐up isolates were obtained. The species isolated and drug susceptibility changed unpredictably during treatment. Of 33 PCC isolates, nine (27.3%) were meticillin resistant. Multiple drug resistance was identified in 41 of 53 (77.3%) MRS isolates from the VMTH and five of nine from the PCC. The sensitivity and specificity of PCR for the detection of meticillin resistance was 34 of 39 (87%) and 86 of 87 (99%), respectively. Risk factors for meticillin resistance for both sites were antibiotic treatment within the last year (P = 0.001), and for VMTH, hospitalization of dogs within the last year (P = 0.001).

Conclusions and clinical importance –  The prevalence of meticillin resistance was not different between VMTH and PCC isolates (P = 0.29). Previous antimicrobial therapy was an important risk factor for the isolation of MRS at both sites.
The effect of ketoconazole on whole blood and skin ciclosporin concentrations in dogs
Authors: Gray Laura L. , Hillier Andrew , Cole Lynette K. , Rajala‐Schultz Päivi J.
Published: January 2013

Background –  Ciclosporin (CSA) is approved for the treatment of canine atopic dermatitis. Ciclosporin is metabolized by liver cytochrome P450 enzymes, a process inhibited by ketoconazole (KTZ).

Hypothesis/Objectives –  The aims of this study were to determine skin and blood CSA concentrations when CSA was administered alone at 5.0 (Treatment 1) or 2.5 mg/kg (Treatment 2) and when CSA was administered at 2.5 mg/kg concurrently with KTZ at 5 (Treatment 3) or 2.5 mg/kg (Treatment 4). We hypothesized that skin and blood CSA concentrations in Treatment 1 would not differ from those obtained with T3 or T4.

Animals –  In a randomized cross‐over study, six healthy research dogs received each of the treatments (Treatment 1, 2, 3 and 4) once daily for 7 days.

Methods –  After the first, fourth and seventh dose for each treatment, a peak and trough skin punch biopsy sample and whole blood sample were collected and analysed with high‐performance liquid chromatography–tandem mass spectrometry. Data were analysed using a repeated measures approach with PROC MIXED in SAS. Pairwise comparisons were performed with least squares means and Tukey–Kramer adjustment for multiple comparisons.

Results –  Mean blood CSA concentrations in Treatment 1 were not different from those in Treatment 2 or 4, but were less than in Treatment 3. Mean skin CSA concentrations in Treatment 1 were greater than in Treatment 2, not different from those in Treatment 4, and less than those in Treatment 3.

Conclusions and clinical importance –  Administration of CSA and KTZ concurrently at 2.5 mg/kg each may be as effective as CSA alone at 5.0 mg/kg for treatment of canine atopic dermatitis.
Fixing the skin barrier: past, present and future – man and dog compared
Authors: Marsella Rosanna
Published: January 2013

Skin barrier dysfunction exists in both human and canine atopic dermatitis, leading to increased water loss and potentially facilitating allergen penetration and sensitization. Both lipid (e.g. ceramides) and protein (e.g. filaggrin) abnormalities have been described. Some are genetically inherited (e.g. filaggrin mutations are one of the major risk factors in humans) and some are secondary and linked to inflammation.

In humans, numerous studies have shown efficacy of emollients and moisturizers in barrier restoration, and this approach has been for years the mainstay of therapy. Recently, this strategy has shown promise as a preventative function.

In veterinary medicine, evidence regarding skin barrier impairment is rapidly building. Decreased ceramides and filaggrin (in some subsets of dogs) have been described. Altered metabolism of ceramides has also been proposed. Despite these preliminary data and the availability of products marketed to improve the skin barrier, evidence regarding the clinical benefit of skin repair intervention is still limited. Preliminary studies have demonstrated that topical application of fatty acids and ceramides and systemic administration of fatty acids improve lipid deficiencies in the skin of dogs with atopic dermatitis, but limited clinical evidence exists.

Disease remission in humans is paralleled by an improved skin barrier, both with calcineurin inhibitors and glucocorticoids. In veterinary medicine, a preliminary study on ciclosporin and prednisone failed to detect significant improvement of water loss, while successful immunotherapy correlated with an improved skin barrier. Controlled, large studies are needed to address the question of which skin repair approach is clinically most effective and whether this can be used as a preventative strategy.
The canine and feline skin microbiome in health and disease
Authors: Weese J. Scott
Published: January 2013

The skin harbours a diverse and abundant, yet inadequately investigated, microbial population. The population is believed to play an important role in both the pathophysiology and the prevention of disease, through a variety of poorly explored mechanisms.

Early studies of the skin microbiota in dogs and cats reported a minimally diverse microbial composition of low overall abundance, most probably as a reflection of the limitations of testing methodology. Despite these limitations, it was clear that the bacterial population of the skin plays an important role in disease and in changes in response to both infectious and noninfectious diseases.

Recent advances in technology are challenging some previous assumptions about the canine and feline skin microbiota and, with preliminary application of next‐generation sequenced‐based methods, it is apparent that the diversity and complexity of the canine skin microbiome has been greatly underestimated. A better understanding of this complex microbial population is critical for elucidation of the pathophysiology of various dermatological (and perhaps systemic) diseases and to develop novel ways to manipulate this microbial population to prevent or treat disease.