The use of scanning electron microscopy and energy dispersive X-ray spectroscopy in a case of occupational death.

Scanning electron microscopy is a technique that provides high-resolution images at the micro- and nano-scale. The combination of scanning electron microscopy and energy dispersive X-ray spectroscopy analysis is developing fast for application in forensic science. In this work, we report a case of work-related traumatic death of a 50-year-old man. The autopsy showed cranial fractures with cerebral haemorrhage. It was more difficult to understand the accident dynamics because the body had been shifted from the accident site to mask what had really taken place. Scanning electron microscopy/energy dispersive X-ray spectroscopy was used to identify the material of the impacting tool and to establish the possible legal responsibility of the employer. In this study, we demonstrate that scanning electron microscopy/energy dispersive X-ray spectroscopy is a useful forensic tool for the analysis of biological samples. Further, for studying the lacerations on the corpse from doubtful blunt tools, scanning electron microscopy/energy dispersive X-ray spectroscopy can assist in demonstrating that the scene has been falsified, as it was in this case.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Different insulin concentrations in resuspended vs. unsuspended NPH insulin: Practical aspects of subcutaneous injection in patients with diabetes.

AIMS: This study measured the insulin concentration (Ins[C]) of NPH insulin in vials and cartridges from different companies after either resuspension (R+) or not (R-; in the clear/cloudy phases of unsuspended NPH). METHODS: Measurements included Ins[C] in NPH(R+) and in the clear/cloudy phases of NPH(R-), and the time needed to resuspend NPH and time for NPH(R+) to separate again into clear/cloudy parts. RESULTS: In vials of NPH(R+) (assumed to be 100%), Ins[C] in the clear phase of NPH(R-) was<1%, but 230±41% and 234±54% in the cloudy phases of Novo Nordisk and Eli Lilly NPH, respectively. Likewise, in pen cartridges, Ins[C] in the clear phase of NPH(R-) was<1%, but 182±33%, 204±22% and 229±62% in the cloudy phases of Novo, Lilly and Sanofi NPH. Time needed to resuspend NPH (spent in tipping) in vials was brief with both Novo (5±1s) and Lilly NPH (6±1s), but longer with all pen cartridges (50±8s, 40±6s and 30±4s from Novo, Lilly and Sanofi, respectively; P=0.022). Time required for 50% separation into cloudy and clear parts of NPH was longer with Novo (60±7min) vs. Lilly (18±3min) in vials (P=0.021), and affected by temperature, but not by the different diameter sizes of the vials. With pen cartridges, separation into clear and cloudy parts was significantly faster than in vials (P<0.01). CONCLUSION: Ins[C] in NPH preparations varies depending on their resuspension or not. Thus, subcutaneous injection of the same number of units of NPH in patients with diabetes may deliver different amounts of insulin depending on its prior NPH resuspension.

An Overview of Lipid Droplets in Cancer and Cancer Stem Cells.

For decades, lipid droplets have been considered as the main cellular organelles involved in the fat storage, because of their lipid composition. However, in recent years, some new and totally unexpected roles have been discovered for them: (i) they are active sites for synthesis and storage of inflammatory mediators, and (ii) they are key players in cancer cells and tissues, especially in cancer stem cells. In this review, we summarize the main concepts related to the lipid droplet structure and function and their involvement in inflammatory and cancer processes.

Glargine metabolism over 24 h following its subcutaneous injection in patients with type 2 diabetes mellitus: a dose-response study.

BACKGROUND AND AIMS: After subcutaneous injection insulin glargine is rapidly metabolized to M1 and M2. In vitro, both M1 and M2 have metabolic effects and bind to IGF-1R similarly to human insulin, whereas glargine exhibits a higher affinity for the IGF-1R and greater mitogenetic effects. The present study was specifically designed to establish the dose-response metabolism of glargine over 24 h following s.c. injection in T2DM subjects on long-term use of glargine. METHODS AND RESULTS: Ten subjects with T2DM were studied during 24 h after s.c. injection of 0.4 (therapeutic) and 0.8 (high dose) U/kg of glargine on two separate occasions during euglycaemic clamps (cross-over design). Glargine, M1 and M2 over 24 h period were determined in appropriately processed plasma samples by a specific liquid chromatography-tandem mass spectrometry assay. Plasma M1 concentration (AUC0-24 h) was detected in all subjects and increased by increasing the glargine dose from therapeutic to high dose (p = 0.008). Glargine was detectable in 6 (therapeutic dose) and 9 (high dose) out of the 10 subjects and also increased by increasing the dose (p = 0.031). However, glargine concentration (AUC0-24 h--high dose) represented at most only 9.7% (4.6-15%) of the total amount of insulin measured in the blood. M2 was not detected at all. CONCLUSION: In T2DM people on long-term use of insulin glargine, even with higher doses (0.8 U/kg), glargine is nearly totally metabolized to the active metabolite M1. Glargine is often detectable in plasma, but its concentration remains well below that needed in vitro to potentiate IGF-1R binding and mitogenesis.

Water soluble nanoporous nanoparticle for in vivo targeted drug delivery and controlled release in B cells tumor context.

Multitasking nanoparticles are gaining great attention for smart drug delivery systems. The exploration of the nano-scale opens new concrete opportunities for revealing new properties and undiscovered cell-particle interactions. Here we present a biodegradable nanoporous silicon nanoparticle that can be successfully employed for in vivo targeted drug delivery and sustained release. The bare nanoporous nanocarriers can be accurately designed and fabricated with an effective control of porosity, surface chemistry and particle size, up to a few nm. The proposed nanoparticles exhibit several remarkable features including high payload, biodegradability, no toxicity, and multiple loading in water without the need of additional chemical reagents at room temperature. The targeting strategy is based on phage display technology that was successfully used to discover cell surface binding peptide for murine B lymphoma A20 cell line. The peptide used in combination with the nanoporous nanoparticles allows an efficient in vivo targeting, a sustained release and a sensible therapeutic effect.

Observation of coherence and partial decoherence of quantized spin waves in nanoscaled magnetic ring structures.

Experiments and simulations are reported, which demonstrate the influence of partial decoherence of spin-wave modes on the dynamics in small magnetic structures. Microfocus Brillouin light scattering spectroscopy was performed on 15 nm thick Ni81Fe19 rings with diameters from 1 to 3 microm. For the so-called "onion" magnetization state several effects were identified. First, in the pole regions of the rings spin-wave wells are created due to the inhomogeneous internal field leading to spin-wave confinement. Second, in the regions in between, modes are observed which show a well pronounced quantization in radial direction but a transition from partial to full coherency in azimuthal direction as a function of decreasing ring size. In particular for larger rings a continuous frequency variation with position is observed which is well reproduced by spin-wave calculations and micromagnetic simulations.

MICA gene polymorphism in the pathogenesis of type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is a typical autoimmune disease and results from the destruction of insulin-producing beta cells of the pancreas. It develops in the presence of genetic susceptibility, even though more than 85% of patients with T1DM do not have a close relative with the disorder. The etiology of T1DM is complex, and both genetic and environmental factors play important roles. A permissive genetic background is required for the development of the islet autoimmune process. The strongest genetic association idengified is that with HLA class II genes located on the short arm of chromosome 6. It is well known that both HLA DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) and DRB1*03-DQA1*0501-DQB1*0201 (DR3-DQ2) are positively, and DRB1*15-DQA1*0102-DQB1*0602 is negatively, associated with T1DM. However, only a minority of the subjects carrying the high-risk haplotypes/genotypes develops the disease, which suggests that additional genes play a crucial role in conferring either protection or susceptibility to T1DM. Major histocompatibility complex (MHC) class I chain-related A (MICA) is located in a candidate susceptibility region and activates natural killer (NK) cells, T cells and gammadelta CD8 T cells by its receptor NKG2D. The polymorphism of the MICA gene is associated with T1DM in different populations as demonstrated in several papers published in the last 7 years.

Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse.

AIMS/HYPOTHESIS: Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. METHODS: VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. RESULTS: Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. CONCLUSIONS/INTERPRETATION: Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

Low dose (1 microg) ACTH test in the evaluation of adrenal dysfunction in pre-clinical Addison's disease.

OBJECTIVE: The presence of 21-hydroxylase autoantibodies (21OHAb) is a marker of adrenal autoimmunity and can be used to identify subjects with pre-clinical Addison's disease. The low-dose (1 microg) ACTH test (LDT) is more sensitive than the high-dose (250 microg) test (HDT) for the diagnosis of pituitary adrenal insufficiency, but no information is available on the use of a LDT in subjects with autoimmune adrenalitis and primary adrenal insufficiency. The aim of our study was to evaluate the clinical use of the LDT in the diagnosis of early adrenocortical dysfunction in patients with adrenal autoantibodies. DESIGN AND METHODS: Firstly, we evaluated the cortisol responses to both a LDT and a HDT in a group of 12 healthy volunteers. We then performed a LDT in 11 subjects positive for 21OHAb, but without clinical signs of Addison's disease identified by screening 920 patients with one or more organ-specific autoimmune diseases. In all cases, the LDT was followed by a sequential HDT which was used as a control test of the sensitivity and specificity of the LDT. RESULTS: In healthy subjects, the peak cortisol levels after the LDT were similar to those after the classical HDT. In 21OHAb-positive subjects, the LDT showed a pathological response in five out of 11 (45%) cases and the diagnostic concordance between the results of the LDT and those of the HDT was 100%. All the five cases with pathological LDT were also positive for adrenal cortex autoantibodies (ACA) and 4/5 had high levels of basal ACTH. One subject with pathological LDT developed clinical Addison's disease 4 months after the test had been performed. CONCLUSIONS: Our study demonstrates that the low dose ACTH test has a high diagnostic sensitivity and specificity for primary adrenal insufficiency and suggests that it can accurately identify subjects with pre-clinical adrenal dysfunction.

Autoantibody recognition of COOH-terminal epitopes of GAD65 marks the risk for insulin requirement in adult-onset diabetes mellitus.

Some type 2 diabetic subjects develop secondary failure to sulphonylurea treatment and require insulin therapy. To test the diagnostic sensitivity and specificity of epitopes of GAD65 autoantibodies (GAD65Ab) for insulin requirement, in patients with latent autoimmune diabetes of the adult, we studied 569 adult subjects with a clinical diagnosis of type 2 diabetes mellitus. All the patients had been initially treated with hypoglycemic agents and/or diet for at least 1 yr. The presence of GAD65Ab (61/569, 10.7%) depended on insulin therapy (P<0.0001), low BMI (P<0.0001), and low basal C-peptide (P = 0.01). The majority of GAD65Ab-positive subjects (47/61, 77%) had antibodies directed to both middle (GAD65-MAb) and COOH-terminal (GAD65-CAb) epitopes. However, GAD65-CAb were more frequent in insulin-treated subjects (92% of GAD65Ab+ individuals) than in subjects treated with hypoglycemic agents and/or diet (18.2% of GAD65Ab+ individuals), while the exclusive presence of GAD65-MAb was more frequent in subjects treated with hypoglycemic agents and/or diet (81.8% vs. 8%) (P<0.0001). The presence of GAD65-CAb had a diagnostic specificity for insulin requirement as high as 99.4% (compared with 96.9% of GAD65Ab as measured in the traditional radiobinding assay) and identified a subgroup of patients with low BMI, low basal C-peptide values, and a need for insulin therapy. Subjects carrying only GAD65-MAb were phenotypically indistinguishable from GAD65Ab-negative patients. Patients positive for GAD65-M+CAb, but not those positive for GAD65-MAb only, showed an increased risk for thyroid autoimmunity, as revealed by the presence of thyroid peroxidase autoantibodies. Our study demonstrates that the use of epitope-specific antibody assays improves the diagnostic specificity of GAD65Ab, and that the presence of GAD65Ab binding to COOH-terminal epitopes is strongly associated with a need for insulin requirement.

HIV infection of the adult thymus: an even more conventional theory explaining CD4 cell decrease and CD8 cell increase in AIDS.

CD4 cells are known to drop and CD8 cells are known to increase in patients with HIV infection. It is accepted that this is the result of HIV selectively killing CD4 cells; we propose an alternative explanation for this phenomenon.