Case Report: Insulin hypersensitivity in youth with type 1 diabetes.

Objective: Immediate type I, type III, and delayed type IV hypersensitivity reactions to insulin are rare, but potentially serious complications of exogenous insulin administration required for the treatment of type 1 diabetes (T1D). Methods: We present four cases of insulin hypersensitivity reactions occurring in youth with T1D and a literature review of this topic. Results: Insulin hypersensitivity reactions included types I, III, and IV with presentations ranging from localized urticaria, erythematous nodules, and eczematous plaques to anaphylaxis with respiratory distress. Reactions occurred in youth with newly diagnosed T1D and in those with long-standing T1D who were using both injection and insulin pump therapy. Multidisciplinary care involving pediatric endocrinology and allergy/immunology utilizing trials of many adjunct therapies yielded minimal improvement. Despite the use of various treatments, including antihistamines, topical therapies, immunosuppressant medications, desensitization trials, and intravenous immune globulin, cutaneous reactions, elevated hemoglobin A1c levels, and negative effects on quality of life remain persistent challenges. One patient became one of the youngest pancreas transplant recipients in the world at age 12 years due to uncontrollable symptoms and intolerable adverse effects of attempted therapies. Conclusion: Although rare, insulin hypersensitivity reactions negatively affect glycemic control and quality of life. These cases demonstrate the varying severity and presentation of insulin hypersensitivity reactions along with the limited success of various treatment approaches. Given the life-sustaining nature of insulin therapy, further studies are needed to better understand the underlying pathophysiology of insulin hypersensitivity and to develop targeted treatment approaches.

Combined autofluorescence and diffuse reflectance for brain tumour surgical guidance: initial ex vivo study results.

This ex vivo study was conducted to assess the potential of using a fibre optic probe system based on autofluorescence and diffuse reflectance for tissue differentiation in the brain. A total of 180 optical measurements were acquired from 28 brain specimens (five patients) with eight excitation and emission wavelengths spanning from 300 to 700 nm. Partial least square-linear discriminant analysis (PLS-LDA) was used for tissue discrimination. Leave-one-out cross validation (LOOCV) was then used to evaluate the performance of the classification model. Grey matter was differentiated from tumour tissue with sensitivity of 89.3% and specificity of 92.5%. The variable importance in projection (VIP) derived from the PLS regression was applied to wavelengths selection, and identified the biochemical sources of the detected signals. The initial results of the study were promising and point the way towards a cost-effective, miniaturized hand-held probe for real time and label-free surgical guidance.

Comparison of Surgical Smoke Generated During Electrosurgery with Aerosolized Particulates from Ultrasonic and High-Speed Cutting.

"Surgical smoke" is an airborne by-product of electrosurgery comprised of vapour and suspended particles. Although concerns exist that exposure may be harmful, there is a poor understanding of the smoke in terms of particle size, morphology, composition and biological viability. Notably, it is not known how the biological tissue source and cutting method influence the smoke. The objective of this study was to develop a collection method for airborne by-product from surgical cutting. This would enable comprehensive analyses of the particulate burden, composition and biological viability. The method was applied to compare the electrosurgical smoke generated (in the absence of any evacuation mechanism) with the aerosolized/airborne by-products generated by ultrasonic and high-speed cutting, from bone and liver tissue cutting. We report a wide range of particle sizes (0.93-806.31 µm for bone, 0.05-1040.43 µm for liver) with 50% of the particles being <2.72 µm (~PM2.5) and 90% being <10 µm (PM10). EDX and biochemical analysis reveal components of biological cells and cellular metabolic activity in particulate from liver tissue cut by electrosurgery and ultrasonic cutting. We show for the first time however that bone saws and ultrasonic cutting do not liberate viable cells from bone.

Unique oestrogen receptor ligand-binding domain sequence of native parrots: a possible link between phytoestrogens and breeding success.

The New Zealand (NZ) native parrots kakapo, kaka and kea are classified as critically endangered, endangered and vulnerable respectively. Successful reproduction of kakapo and kaka is linked to years of high levels of fruiting in native flora (mast years). To assess a possible hormonal link between native plants and reproductive success in these parrots in mast years, we examined the ligand-binding domains (LBD) of the progesterone receptor (PR), androgen receptor (AR), estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) in NZ native (kakapo, kaka, kea and kakariki) and non-native (Australian cockatiel) parrots and compared them with those in the chicken. The amino acid sequences for PR, AR, ESR1 and ESR2 shared >90% homology among the NZ parrots, the cockatiel and, in most cases, the chicken. The exception was for the ESR1 LBD, which contained an extra eight amino acids at the C-terminal in all the parrots compared with the chicken and with published sequences of non-parrot species. These results support the notion that the ESR1 LBD of parrots responds differently to putative oestrogenic compounds in native trees in NZ during times of intermittent masting. In turn, this may provide important information for generating parrot-specific bioassays and linkages to steroidogenic activity in native plants.

Spatially offset Raman spectroscopy for photon migration studies in bones with different mineralization levels.

The ability of Spatially Offset Raman Spectroscopy (SORS) to obtain chemically specific information from below the sample surface makes it a promising technique for non-invasive in vivo diagnosis of bone conditions by sampling bone through the skin. The depth below a surface interrogated by SORS depends on the system's optical properties and is difficult to estimate for complex bone material. This paper uses 830 nm laser excitation to investigate the influence of bone mineralization on photon migration properties in deer antler cortex, equine metacarpal cortex and whale tympanic bulla. Thin slices form each type of bone (thickness: 0.6-1.0 mm) were cut and put together on top of each other forming stacks with a total thickness of 4.1-4.7 mm. A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as a test material for Raman signal recovery and was placed in between the individual bone slices within the stack. At SORS offsets of 8.0-9.5 mm Raman bands of materials not present in healthy bone (e.g. PTFE as an example) can be recovered through 4.4-4.7 mm of cortical bone tissue, depending on mineralization level and porosity. These findings significantly increase our understanding of SORS analysis through bones of different composition and provide information that is vital to determine the value of SORS as a medical diagnostic technique. The data serve to define which SORS offset is best deployed for the non-invasive detection of chemically specific markers associated with infection, degeneration and disease or cancer within bone.

Single-stranded RNA viruses infecting the invasive Argentine ant, Linepithema humile.

Social insects host a diversity of viruses. We examined New Zealand populations of the globally widely distributed invasive Argentine ant (Linepithema humile) for RNA viruses. We used metatranscriptomic analysis, which identified six potential novel viruses in the Dicistroviridae family. Of these, three contigs were confirmed by Sanger sequencing as Linepithema humile virus-1 (LHUV-1), a novel strain of Kashmir bee virus (KBV) and Black queen cell virus (BQCV), while the others were chimeric or misassembled sequences. We extended the known sequence of LHUV-1 to confirm its placement in the Dicistroviridae and categorised its relationship to closest relatives, which were all viruses infecting Hymenoptera. We examined further for known viruses by mapping our metatranscriptomic sequences to all viral genomes, and confirmed KBV, BQCV, LHUV-1 and Deformed wing virus (DWV) presence using qRT-PCR. Viral replication was confirmed for DWV, KBV and LHUV-1. Viral titers in ants were higher in the presence of honey bee hives. Argentine ants appear to host a range of' honey bee' pathogens in addition to a virus currently described only from this invasive ant. The role of these viruses in the population dynamics of the ant remain to be determined, but offer potential targets for biocontrol approaches.

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review.

The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein- and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>€35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robustness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations, and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.

Raman Spectroscopy of Blood and Blood Components.

Blood is a bodily fluid that is vital for a number of life functions in animals. To a first approximation, blood is a mildly alkaline aqueous fluid (plasma) in which a large number of free-floating red cells (erythrocytes), white cells (leucocytes), and platelets are suspended. The primary function of blood is to transport oxygen from the lungs to all the cells of the body and move carbon dioxide in the return direction after it is produced by the cells' metabolism. Blood also carries nutrients to the cells and brings waste products to the liver and kidneys. Measured levels of oxygen, nutrients, waste, and electrolytes in blood are often used for clinical assessment of human health. Raman spectroscopy is a non-destructive analytical technique that uses the inelastic scattering of light to provide information on chemical composition, and hence has a potential role in this clinical assessment process. Raman spectroscopic probing of blood components and of whole blood has been on-going for more than four decades and has proven useful in applications ranging from the understanding of hemoglobin oxygenation, to the discrimination of cancerous cells from healthy lymphocytes, and the forensic investigation of crime scenes. In this paper, we review the literature in the field, collate the published Raman spectroscopy studies of erythrocytes, leucocytes, platelets, plasma, and whole blood, and attempt to draw general conclusions on the state of the field.

Raman spectroscopy as a novel tool for monitoring biochemical changes and inter-donor variability in stored red blood cell units.

Individual units of donated red blood cells (RBCs) do not ordinarily undergo analytical testing prior to transfusion. This study establishes the utility of Raman spectroscopy for analyzing the biochemistry of stored RBC supernatant and reveals interesting storage-related changes about the accumulation of lactate, a chemical species that may be harmful to certain patients. The data show measurable variations in supernatant composition and demonstrate that some units of donated RBCs accumulate lactate much more readily than others. The spectra also indicate a higher relative concentration of lactate in units collected from male donors than female donors (p = 0.004) and imply that there is a greater degree of variability at later stages of storage in units from older male donors (>45 years). The study proves that Raman analysis has promise for elucidating the relationship between the metabolism of stored RBCs and donor characteristics. It also suggests that there may be benefit in developing a Raman instrument for the rapid non-invasive assessment of blood-bag biochemistry by measuring through plastic over-layers.

Is the Collagen Primed for Mineralization in Specific Regions of the Turkey Tendon? An Investigation of the Protein-Mineral Interface Using Raman Spectroscopy.

The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm(-1) increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease.

Accelerated Unilateral Radiographic Huntingtonian Changes Following Neoadjuvant Chemotherapy for a Nongerminomatous Germ Cell Tumor Leading to Identification of Occult Disease in the Dorsal Striatum.

Basal ganglia nongerminomatous germ cell tumors comprise 10% to 15% of germ cell tumor and have substantial morbidity at the time of local failure. In this submitted image we present a case where neoadjuvant chemotherapy unmasked a unilateral caudate head loss consistent with Huntingtonian changes. Careful review of the patient's imaging identified disease within the dorsal striatum that was not previously identified at the time of diagnosis. Review of the diffusion tensor fractional anisotropy imaging identified progressive white matter likely secondary to the occult disease within the dorsal striatum. Although this patient was asymptomatic and had no signs of a movement disorder, similar findings have been noted to be a prelude to such findings several months later. The occult disease was incorporated into the patient's radiotherapy planning target volume as oversight of these changes would have led to a marginal miss and potential early disease relapse.

Towards the in vivo prediction of fragility fractures with Raman spectroscopy.

Fragility fractures, those fractures which result from low level trauma, have a large and growing socio-economic cost in countries with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture because both phases contribute to bone's mechanical properties. In this study we analyse excised bone with Raman spectroscopy and multivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excised work that on average, bone fragments from the necks of fractured femora are more mineralised (by 5-10%) than (cadaveric) non-fractured controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy and diseased human cohorts), we identify methodological developments which will be used to improve the statistical significance of future experiments and may eventually lead to more sensitive prediction of fragility fractures. © 2015 The Authors. Journal of Raman Spectroscopy Published by John Wiley & Sons, Ltd.

Functional adaptation of long bone extremities involves the localized "tuning" of the cortical bone composition; evidence from Raman spectroscopy.

In long bones, the functional adaptation of shape and structure occurs along the whole length of the organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ's length. Raman spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10% toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses, sheep, and deer) gave similar results with bone material composition changing across tens of centimeters. The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions about the biochemical control mechanisms involved. In addition to their biological interest, the data provide timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring bone composition in vivo (particularly with regard to sampling and measurement protocol).

Millimeter-scale mapping of cortical bone reveals organ-scale heterogeneity.

Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the phosphate-to-amide I ratio (a measure of the mineral to collagen ratio) can vary by as much as 8%, and the phosphate-to-carbonate ratio (a measure of carbonate inclusion in mineral crystals) by as much as 5%. The data are preliminary but are important because they reveal a spatial variation at a scale that is much larger than many of the spectral maps reported in the literature to date. Thus they illustrate natural variation in chemical composition that could have been overlooked in such studies or could have appeared as an undue error where the overall composition of the bone was investigated. Quantifying the variation in mid-shaft cortical bone at the millimeter/centimeter scale reduces the possibility of natural heterogeneity obscuring the average bone composition, or being mistaken for experimental signal, and results in an improvement in the sampling accuracy analogous to that obtained by switching from micrometer-size point spectra of bones to spectral images obtained across hundreds of micrometers. Although the study was carried out using Raman spectroscopy, the underlying cause of the variation is ascribed to the variation of the chemical composition of the bone; therefore the findings have direct implications for other chemically specific analytical methods such as Fourier transform infrared spectroscopy or nuclear magnetic resonance spectroscopy.

Evidence from Raman spectroscopy of a putative link between inherent bone matrix chemistry and degenerative joint disease.

OBJECTIVE: Osteoarthritis (OA) is a common debilitating disease that results in degeneration of cartilage and bone in the synovial joints. Subtle changes in the molecular structure of the subchondral bone matrix occur and may be associated with cartilage changes. The aim of this study was to explore whether the abnormal molecular changes observed in the matrix of OA subchondral bone can be identified with Raman spectroscopy. METHODS: Tibial plateaus from patients undergoing total knee replacement for OA (n = 10) were compared with healthy joints from patients undergoing leg amputation (n = 5; sex- and laterality-matched) and with non-OA cadaveric knee specimens (n = 5; age-matched). The samples were analyzed with Raman spectroscopy, peripheral quantitative computed tomography, and chemical analysis to compare changes in defined load-bearing sites in both the medial and lateral compartments. RESULTS: OA subchondral bone matrix changes were detected by Raman spectroscopy. Within each cohort, there was no spectral difference in bone matrix chemistry between the medial and lateral compartments, whereas a significant spectral difference (P < 0.001) was observed between the non-OA and OA specimens. Type I collagen chain ratios were normal in the non-OA specimens but were significantly elevated in the OA specimens. CONCLUSION: In comparing the results of Raman spectroscopy with those obtained by other standard techniques, these findings show, for the first time, that subchondral bone changes, or inherent differences, exist in both the medial and lateral (beneath intact cartilage) compartments of OA knees. The development of Raman spectroscopy as a screening tool, based on molecular-specific modifications in bone, would facilitate the identification of clinical disease, including early molecular changes.

Recirculating bone marrow B cells in C57BL/6 mice are more tolerant of highly hydrophobic and highly charged CDR-H3s than those in BALB/c mice.

To test whether mechanisms controlling the range of diversity of the developing antibody repertoire in C57BL/6 mice (IgH(b)) operate similarly to those identified in BALB/c mice (IgH(a)), we compared the sequences of VH 7183-containing H-chain transcripts from sorted adult bone marrow C57BL/6 B-cell subsets with those previously obtained from BALB/c mice. Patterns of VDJ gene segment utilization and CDR-H3 amino acid composition, charge, and average length in C57BL/6 pro-B cells were similar, although not identical, to BALB/c pro-B cells. However, C57BL/6 mature, recirculating B cells failed to demonstrate the reduction in the use of VH81X and the narrowing in the range of variance of CDR-H3 hydrophobicity that characterizes B-cell maturation in BALB/c mice. To further test the ability of the C57BL/6 strain to discard B cells expressing highly charged CDR-H3s, we introduced a mutant IgH(a) DH allele that forces use of arginine, asparagine, and histidine. Unlike BALB/c mice, C57BL/6 mice congenic for the charged DH maintained normal numbers of mature, recirculating B cells that were enriched for charged CDR-H3s. Together these findings indicate that the mature C57BL/6 B-cell pool permits expression of immunoglobulins with antigen-binding sites that are typically discarded during late-stage bone marrow B-cell development in BALB/c mice.

Dynamics of the last glacial maximum Antarctic ice-sheet and its response to ocean forcing.

Retreat of the Last Glacial Maximum (LGM) Antarctic ice sheet is thought to have been initiated by changes in ocean heat and eustatic sea level propagated from the Northern Hemisphere (NH) as northern ice sheets melted under rising atmospheric temperatures. The extent to which spatial variability in ice dynamics may have modulated the resultant pattern and timing of decay of the Antarctic ice sheet has so far received little attention, however, despite the growing recognition that dynamic effects account for a sizeable proportion of mass-balance changes observed in modern ice sheets. Here we use a 5-km resolution whole-continent numerical ice-sheet model to assess whether differences in the mechanisms governing ice sheet flow could account for discrepancies between geochronological studies in different parts of the continent. We first simulate the geometry and flow characteristics of an equilibrium LGM ice sheet, using pan-Antarctic terrestrial and marine geological data for constraint, then perturb the system with sea level and ocean heat flux increases to investigate ice-sheet vulnerability. Our results identify that fast-flowing glaciers in the eastern Weddell Sea, the Amundsen Sea, central Ross Sea, and in the Amery Trough respond most rapidly to ocean forcings, in agreement with empirical data. Most significantly, we find that although ocean warming and sea-level rise bring about mainly localized glacier acceleration, concomitant drawdown of ice from neighboring areas leads to widespread thinning of entire glacier catchments-a discovery that has important ramifications for the dynamic changes presently being observed in modern ice sheets.

Pediatric genitourinary tumors.

PURPOSE OF REVIEW: We will review the 2010/2011 literature on pediatric genitourinary tumors and highlight the most significant publications. RECENT FINDINGS: New techniques such as gene expression profiling, PET, and nephron-sparing surgery are being incorporated into contemporary treatments for pediatric patients with genitourinary tumors. Biologic markers are increasingly being used to help with risk stratification of patients and to identify new targets for therapy. WT1 mutation and 11p15 loss of heterozygosity have been associated with relapse in very low-risk Wilms tumors treated with surgery alone and may help reduce the use of chemotherapy in some children. Meta-analysis of data on the use of high-dose chemotherapy with autologous hematopoietic stem cell rescue in patients with relapsed Wilms tumor and rhabdomyosarcoma suggests that some patients may benefit more from conventional salvage chemotherapy. New agents are needed for patients with high-risk and relapsed disease to improve outcomes. SUMMARY: In general, the prognosis for patients with pediatric genitourinary tumors is favorable. Further understanding of the biology in these tumors is helping to determine risk stratification, treatment strategies, and candidates for new drug development.

Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein.

This study describes the application of quality by design (QbD) principles to the development and implementation of a major manufacturing process improvement for a commercially distributed therapeutic protein produced in Chinese hamster ovary cell culture. The intent of this article is to focus on QbD concepts, and provide guidance and understanding on how the various components combine together to deliver a robust process in keeping with the principles of QbD. A fed-batch production culture and a virus inactivation step are described as representative examples of upstream and downstream unit operations that were characterized. A systematic approach incorporating QbD principles was applied to both unit operations, involving risk assessment of potential process failure points, small-scale model qualification, design and execution of experiments, definition of operating parameter ranges and process validation acceptance criteria followed by manufacturing-scale implementation and process validation. Statistical experimental designs were applied to the execution of process characterization studies evaluating the impact of operating parameters on product quality attributes and process performance parameters. Data from process characterization experiments were used to define the proven acceptable range and classification of operating parameters for each unit operation. Analysis of variance and Monte Carlo simulation methods were used to assess the appropriateness of process design spaces. Successful implementation and validation of the process in the manufacturing facility and the subsequent manufacture of hundreds of batches of this therapeutic protein verifies the approaches taken as a suitable model for the development, scale-up and operation of any biopharmaceutical manufacturing process.