Diabetic Foot Complications and Their Risk Factors from a Large Retrospective Cohort Study

BackgroundFoot complications are considered to be a serious consequence of diabetes mellitus, posing a major medical and economical threat. Identifying the extent of this problem and its risk factors will enable health providers to set up better prevention programs. Saudi National Diabetes Registry (SNDR), being a large database source, would be the best tool to evaluate this problem.MethodsThis is a cross-sectional study of a cohort of 62,681 patients aged ≥25 years from SNDR database, selected for studying foot complications associated with diabetes and related risk factors.ResultsThe overall prevalence of diabetic foot complications was 3.3% with 95% confidence interval (95% CI) of (3.16%–3.44%), whilst the prevalences of foot ulcer, gangrene, and amputations were 2.05% (1.94%–2.16%), 0.19% (0.16%–0.22%), and 1.06% (0.98%–1.14%), respectively. The prevalence of foot complications increased with age and diabetes duration predominantly amongst the male patients. Diabetic foot is more commonly seen among type 2 patients, although it is more prevalent among type 1 diabetic patients. The Univariate analysis showed Charcot joints, peripheral vascular disease (PVD), neuropathy, diabetes duration ≥10 years, insulin use, retinopathy, nephropathy, age ≥45 years, cerebral vascular disease (CVD), poor glycemic control, coronary artery disease (CAD), male gender, smoking, and hypertension to be significant risk factors with odds ratio and 95% CI at 42.53 (18.16–99.62), 14.47 (8.99–23.31), 12.06 (10.54–13.80), 7.22 (6.10–8.55), 4.69 (4.28–5.14), 4.45 (4.05–4.89), 2.88 (2.43–3.40), 2.81 (2.31–3.43), 2.24 (1.98–2.45), 2.02 (1.84–2.22), 1.54 (1.29–1.83), and 1.51 (1.38–1.65), respectively.ConclusionsRisk factors for diabetic foot complications are highly prevalent; they have put these complications at a higher rate and warrant primary and secondary prevention programs to minimize morbidity and mortality in addition to economic impact of the complications. Other measurements, such as decompression of lower extremity nerves, should be considered among diabetic patients.     

Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2–16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88−M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88−M2e2-16 phages in the presence of incomplete Freund’s adjuvant, induced robust M2e-specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.     

A comparison of the predictive accuracy of three screening models for pulmonary arterial hypertension in systemic sclerosis

IntroductionThere is evidence that early screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) improves outcomes. We compared the predictive accuracy of two recently published screening algorithms (DETECT 2013 and Australian Scleroderma Interest Group (ASIG) 2012) for SSc-associated PAH (SSc-PAH) with the commonly used European Society of Cardiology/European Respiratory Society (ESC/ERS 2009) guidelines.MethodsWe included 73 consecutive SSc patients with suspected PAH undergoing right heart catheterization (RHC). The three screening models were applied to each patient. For each model, contingency table analysis was used to determine sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values for PAH. These properties were also evaluated in an ‘alternate scenario analysis’ in which the prevalence of PAH was set at 10%.ResultsRHC revealed PAH in 27 (36.9%) patients. DETECT and ASIG algorithms performed equally in predicting PAH with sensitivity and NPV of 100%. The ESC/ERS guidelines had sensitivity of 96.3% and NPV of only 91%, missing one case of PAH; these guidelines could not be applied to three patients who had absent tricuspid regurgitant (TR) jet. The ASIG algorithm had the highest specificity (54.5%). With PAH prevalence set at 10%, the NPV of the models was unchanged, but the PPV dropped to less than 20%.ConclusionsIn this cohort, the DETECT and ASIG algorithms out-perform the ESC/ERS guidelines, detecting all patients with PAH. The ESC/ERS guidelines have limitations in the absence of a TR jet. Ultimately, the choice of SSc-PAH screening algorithm will also depend on cost and ease of application.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0517-5) contains supplementary material, which is available to authorized users.     

WildCARDs: Inflammatory caspases directly detect LPS

Inflammasomes are sensors that serve as activation platforms for caspase-1 — a mechanism that set the prevailing paradigm for inflammatory caspase activation. A recent Nature paper by Shi et al. upends this paradigm by describing an unprecedented model for caspase activation whereby caspase-4, -5, and -11 directly bind their agonist, cytosolic LPS, triggering auto-activation and subsequent pyroptotic cell death.The inflammatory caspases — among them caspase-1, murine caspase-11, and human caspase-4 and -5 (homologs of murine caspase-11) — are central to depriving infectious agents of intracellular replication niches. Upon responding to a given stimulus, they become catalytically active and initiate a form of programmed inflammatory cell death termed pyroptosis. By examining their homology and adjacent chromosomal arrangement in humans and other mammals, it is apparent that the inflammatory caspases originate from a series of gene duplications and subsequent divergences.A balanced caspase-1 response is critical to defense against a variety of infectious agents, whereas its aberrant activation underlies a number of immune pathologies. Less is known about caspase-11, -4, and -5 in infection; however, we have shown that one physiological role of caspase-11 is to detect and help clear cytosol invasive infections, such as those caused by Burkholderia thailandensis¹. More recent work has shown that caspase-11 mediates resistance to DSS-induced colitis² and clearance of Salmonella enterica serovar Typhimurium-infected cells in the intestinal epithelium³, perhaps limited to the times when these bacteria enter the cytosol. Likewise, caspase-4 responds to S. Typhimurium, enteropathogenic E. coli³, and Shigella flexneri⁴ infections in human intestinal epithelial cells. As with caspase-1, moderation of caspase-11 activity is key to limiting immune pathology: much of the lethality of bolus lipopolysaccharide (LPS) injection is mediated by caspase-11^5,6,7,8. Shedding light on the mechanisms underlying these observations, our lab and that of Dr Vishva Dixit independently determined that caspase-11 is activated in response to cytosolic LPS^7,8; however, whether caspase-4 (and/or -5) functions similarly was not determined.Of the inflammatory caspases, the activation mechanism of caspase-1 is the best described. Via its N-terminal CARD domain and an adaptor protein called ASC, caspase-1 interacts with a family of cytosolic proteins, the inflammasomes, that detect signatures of infection (Figure 1). It then initiates pyroptosis and directs proinflammatory cytokine secretion. Inflammasomes thus follow the paradigm of apoptotic caspase activation, where apoptosis initiators caspase-2, -8 and -9 are recruited and activated by death domain family-containing upstream sensors: the piddosome, DISC, and apoptosome, respectively. Therefore, we and others assumed that the model of upstream sensor activating downstream caspase would hold for the other inflammatory caspases as well. For example, Kayagaki and colleagues coined the term ''noncanonical inflammasome pathway'' to describe activation of caspase-11 by a putative LPS sensor^5,6. However, a recent elegant paper by Shi et al.⁹ proves this hypothesis wrong and describes an entirely novel paradigm of caspase activation. Moreover, the authors address many of the gaps in our understanding of caspase-11, -4, and -5 biology.Open in a separate windowFigure 1Schematic of canonical and noncanonical inflammasome pathways for inflammatory caspase activation. Left: Inflammasomes such as AIM2, NLRP3, and NLRC4 detect contamination of the cytosol with microbial ligands (e.g., DNA, flagellin, bacterial type 3 secretion system components) or certain cellular perturbations. Via the adaptor protein ASC, they subsequently activate caspase-1 (in the case of certain CARD-containing inflammasomes, such as NLRC4, direct interaction with caspase-1 can also occur), which initiates pyroptosis and secretion of the proinflammatory cytokines IL-1β and IL-18. Right: Caspase-4, -5, and -11 directly bind cytosolic LPS from Gram-negative bacteria. They subsequently oligomerize, activate, and initiate pyroptosis.Using electroporation to deliver bacterial components into the cytosol of cells, the authors first determined that caspase-4 responds to LPS in human monocytes by triggering pyroptosis. These findings extended to non-myeloid cells, where caspase-4 is constitutively expressed. The authors then demonstrated that caspase-4 and caspase-11 are functionally interchangable, supporting that they are homologs.Shi and colleagues next began identifying the molecule that actually binds LPS in the cytosol. They screened a number of NLRs and CARD domain-containing proteins, but no candidates emerged. In agreement with this, unpublished work from our lab also ruled out virtually all known CARD-containing proteins as the LPS sensor. Clues to the identity of the sensor arose from the following astute observations: First, Shi et al. noticed that both caspase-4 and caspase-11, when purified from E. coli, eluted from columns as large oligomers, suggesting activation, whereas they eluted as monomers when expressed in and purified from insect cells. Second, they found that the LPS contents of caspase-4 and -11 purified from E. coli were three orders of magnitude higher than what they typically observed when purifying bacterial proteins. Together, these results suggested that caspase-4 and -11 directly bind LPS. A series of pull-downs and surface plasmon resonance experiments confirmed this notion, revealing stable interaction of LPS with caspase-4 and -11 in cells transfected with LPS. Furthermore, the authors showed that caspase-5 similarly binds LPS. In all cases, LPS binding and caspase oligomerization was CARD domain dependent; indeed, purified caspase-4 and -11 CARD domains were sufficient to bind LPS and oligomerize. Three regions of basic residues in the caspase-11 CARD domain — mostly conserved in caspase-4 and -5, but not caspase-1 — were critical for LPS binding. Last, the authors determined that caspase-11 and -4 oligomerization stimulates activation, as measured by cleavage of a fluorogenic substrate. Interestingly, the known antagonists of caspase-11 activation Lipid IVa and atypical LPS from Rhodobacter sphaeroides bound caspase-4 and -11, but failed to induce oligomerization and activation.The Shi et al. paper brings to light a number of fascinating perspectives. First, binding of LPS by caspase-4, -5, and -11 establishes a new paradigm for caspase activation: Direct detection of a cell death-inducing ligand by a caspase. As the authors noted, this is analogous to horseshoe crab factors C and G, which bind LPS and β-(1,3)-D-glucan, respectively, and initiate coagulation cascades in haemolymphs.Second, the cell expression patterns of caspase-11, -4, and -5 may have important implications in future strategies for treating endotoxemia and Gram-negative sepsis. Caspase-11 expression is inducible in myeloid cells, where its basal expression is low; in contrast, caspase-4 appears to be constitutively expressed in human myeloid cells. Therefore, aberrant translocation of LPS into the cytosol of human myeoloid cells may not require priming to activate caspase-4 and initiate pyroptosis, perhaps sensitizing humans to the deleterious effects of LPS compared to mice. Investigating other cell type expression differences in this context will be informative.In answering so many questions about the biology of the inflammatory caspases, the work of Shi and colleagues raises many more. Among them: Why do antagonists of caspase-11 fail to induce oligomerization? How do the CARD domains of these caspases “see” LPS? During binding of LPS by MD2, the acyl chains of lipid A extend into the binding cleft of MD2¹⁰ in a manner sensitive to acyl chain length; in contrast, caspase-11 detects very diverse lipid A acyl chain lengths and structures, such as those of Salmonella and Legionella species^7,8,11, suggesting that the CARD domain may wrap around the lipid groups of LPS near the phosphate head groups of lipid A. Insights into these questions will surely come from crystal structures of caspase-11, -4, and -5 bound to various LPS structures.     

Chemokine-Like Receptor 1 Deficiency Does Not Affect the Development of Insulin Resistance and Nonalcoholic Fatty Liver Disease in Mice

The adipokine chemerin and its receptor, chemokine-like receptor 1 (Cmklr1), are associated with insulin resistance and nonalcoholic fatty liver disease (NAFLD), which covers a broad spectrum of liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). It is possible that chemerin and/or Cmklr1 exert their effects on these disorders through inflammation, but so far the data have been controversial. To gain further insight into this matter, we studied the effect of whole-body Cmklr1 deficiency on insulin resistance and NAFLD. In view of the primary role of macrophages in hepatic inflammation, we also transplanted bone marrow from Cmklr1 knock-out (Cmklr1^-/-) mice and wild type (WT) mice into low-density lipoprotein receptor knock-out (Ldlr^-/-) mice, a mouse model for NASH. All mice were fed a high fat, high cholesterol diet containing 21% fat from milk butter and 0.2% cholesterol for 12 weeks. Insulin resistance was assessed by an oral glucose tolerance test, an insulin tolerance test, and by measurement of plasma glucose and insulin levels. Liver pathology was determined by measuring hepatic inflammation, fibrosis, lipid accumulation and the NAFLD activity score (NAS). Whole-body Cmklr1 deficiency did not affect body weight gain or food intake. In addition, we observed no differences between WT and Cmklr1^-/- mice for hepatic inflammatory and fibrotic gene expression, immune cell infiltration, lipid accumulation or NAS. In line with this, we detected no differences in insulin resistance. In concordance with whole-body Cmklr1 deficiency, the absence of Cmklr1 in bone marrow-derived cells in Ldlr^-/- mice did not affect their insulin resistance or liver pathology. Our results indicate that Cmklr1 is not involved in the pathogenesis of insulin resistance or NAFLD. Thus, we recommend that the associations reported between Cmklr1 and insulin resistance or NAFLD should be interpreted with caution.     

The Progeroid Phenotype of Ku80 Deficiency Is Dominant over DNA-PKCS Deficiency

Ku80 and DNA-PK_CS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80^−/− mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pk_cs^−/− mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80^−/−, dna-pk_cs^−/− and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PK_CS causes reduced lifespan and bodyweights, which is most severe in ku80^−/− mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80^−/− mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PK_CS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PK_CS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PK_CS are essential for NHEJ.     

EFFECTS OF THE HALOPHYTES TECTICORNIA INDICA AND SUAEDA FRUTICOSA ON SOIL ENZYME ACTIVITIES IN A MEDITERRANEAN SABKHA

In the present work, we studied the effectiveness of the predominant halophytes of Soliman sabkha (Tecticornia indica and Suaeda fruticosa) to promote soil biological activities and ecosystem productivity. Soil Arylsulphatese ARY, β-glucosidase β-GLU, phosphatase PHO, invertase INV, urease URE, and dehydogenase DES activities in Extra- and Intra-tuft halophytes and plant productivity were assessed. Results revealed a high increase of microbial community and ARY, β-GLU, PHO, INV, URE and DES activities (+298%, +400%, +800%, +350%, +320%, +25% and +759%, respectively) in Intra-tuft rhizosphere as compared to Extra-tuft one, which is likely due to the significant decrease of salinity in the rhizosphere of Tecticornia indica and Suaeda fruticosa. Both perennial plants exhibited high productivities (7.4 t dry weight ha^?1 and 2.2 t dry weight ha^?1, respectively) and Na⁺-hyperaccumulating capacities (0.75 t Na⁺ ha^?1 and 0.22 t Na⁺ ha^?1, respectively), reducing salt constraint and favouring soil fertility. This constitutes a promising alternative to enhance productivity in such a salt-affected biotope by offering suitable microhabitat for annual glycophytes.     

Biodegradation of Chlorpyrifos by a Newly Isolated Bacillus subtilis Strain,Y242

A bacterial strain Y242 isolated from agricultural wastewater was found to be highly effective in degrading chlorpyrifos. On the basis of morphology, physiological characteristics, biochemical tests, and phylogenetic analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under different culture conditions formulated according to the Plackett-Burman experimental design. It was observed that B. subtilis Y242 was able to utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing concentration up to 150 mg/L. A growth medium formulated based on the results of the Plackett-Burman experiment and supplied with 150 mg/L chlorpyrifos recorded 95.12% pesticide decomposition within 48 h. Degradation study of chlorpyrifos by B. subtilis Y242 was examined by gas chromatography–mass spectrometer (GC-MS) and high-performance liquid chromatography (HPLC). These results suggest that B. subtilis Y242 will be potentially useful in the cleanup of contaminated pesticide waste in the environment.     

Effect of simultaneous down-regulation of pectate lyase and endo-β-1,4-glucanase genes on strawberry fruit softening

Strawberry is a soft fruit with a short postharvest shelf-life. The loss of fruit firmness during ripening is mainly due to the disassembly of parenchyma cell walls mediated by the expression of genes encoding enzymes acting on pectins, such as pectate lyase, or hemicellulose, e.g. endo-β-1,4-glucanase. To determine if the simultaneous down-regulation of FaplC and FaEG3 genes, encoding a pectate lyase and a endo-β-1,4-glucanase, respectively, exerted an additive effect on strawberry softening, transgenic plants expressing tandem antisense sequences of both genes under the control of the constitutive promoter CaMV35S were generated. Fifteen independent transgenic lines were obtained and fruit yields and several quality parameters of transgenic ripe fruit were recorded during two consecutive years. Fruit yield was reduced in most of the lines, especially in the first evaluation period, and five out of 15 lines (33 %) did not set fruit. The expression of FaplC and FaEG3 genes was measured in ripe fruits from six selected lines showing the highest fruit yields. All selected lines showed a high level of FaplC gene silencing, ranging from 97 to 71 %; however, FaEG3 gene expression was only significantly down-regulated in two lines. Fruit colour and soluble solids contents were similar in control and transgenic ripe fruits, while fruit weight was slightly lower than control in some of the lines. In all lines, transgenic fruits were significantly firmer than control, with an increase in firmness ranging from 19 to 32 %. The reduction of fruit softening in transgenic fruits was not correlated with the suppression of FaEG3 gene expression, and lines with the highest simultaneous down-regulation of FaplC and FaEG3 showed similar fruit firmness to lines where only FaplC was suppressed. These results indicate that pectate lyase and endo-β-1,4-glucanase do not act in an additive or synergistic way during strawberry softening, and question the role of glucanases in this process.     

Nutritional and technological assessment of durum wheat-faba bean enriched flours,and sensory quality of developed composite bread

Faba beans are acknowledged as a good source of proteins, minerals, fibers, vitamins and antioxidants. A blending study was undertaken in order to prepare naturally bread from enriched flours with added nutritional value, mainly in terms of Iron and proteins. Enriched flours were prepared with varied levels (25, 30, 35 and 40%) of whole faba bean flour to assess the effects of this substitution on their nutritional and technological properties. Then, whole durum wheat bread (regular) and enriched bread at 40% substitution level (composite bread) were prepared and subjected to sensory evaluation. The substitution level of composite bread was selected on the basis of Iron and proteins contents and technological results of the flour blends. Nutritionally, except for moisture, fibers, fat, zinc and sodium values, significant (p < 0.05) increases were showed in ash, proteins, minerals, total phenolic compounds, condensed tannins, total flavonoids and anti-radical activity values. Technologically, significant (p < 0.05) decreases were recorded for lightness and whiteness index. The gluten strength value revealed a significant (p < 0.05) decrease as whole faba bean flour was added. On the sensory level, the level of substitution (40%) chosen for the manufacture of composite bread resulted in acceptable bread by consumers. Moreover, composite bread was most preferred in aroma as it imparts a feeling of satiety. The observed nutritional improvements could be useful for malnourished people, including those having Iron and proteins deficiencies. Technologically, the observed changes didn’t present limitations since composite bread was accepted by consumers even at 40% substitution level. Besides, the slight preference of composite bread aroma might encourage its consumption by consumers. Also, its promotion of satiety is important for gluten sensitivity sufferers. Our results suggested that 40% is the appropriate ratio to increase, at the same time, Iron and proteins contents of enriched flours as well as their overall nutritional quality. Also it was possible to produce natural composite bread at this level (40%) while maintaining adequate technological and sensory quality.