Reproducibility of the mfERG between instruments

Purpose First, to examine both the reproducibility of the multifocal electroretinogram (mfERG) recorded on different versions of the same instrument, and the repeatability of the mfERG recorded on a single instrument using two different amplifiers. Second, to demonstrate a means by which multicenter and longitudinal studies that use more than one recording instrument can compare and combine data effectively. Methods Three different amplifiers and two mfERG setups, one using VERIS™ 4.3 software (mfERG1) and another using VERIS™ Pro 5.2 software (mfERG2), were evaluated. A total of 73 subjects with normal vision were tested in three groups. Group 1 (n = 42) was recorded using two amplifiers in parallel on mfERG1. Group 2 (n = 52) was recorded on mfERG2 using a single amplifier. Group 3 was a subgroup of 21 subjects from groups 1 and 2 that were tested sequentially on both instruments. A fourth group of 26 subjects with diabetes were also recorded using the two parallel amplifiers on mfERG1. P1 implicit times and N1-P1 amplitudes of the 103 local first order mfERGs were measured, and the differences between the instruments and amplifiers were evaluated as raw scores and Z-scores based on normative data. Measurements of individual responses and measurements averaged over the 103 responses were analyzed. Results Simultaneous recordings made on mfERG1 with the two different amplifiers showed differences in implicit times but similar amplitudes. There was a mean implicit time difference of 2.5 ms between the amplifiers but conversion to Z-scores improved their agreement. Recordings made on different days with the two instruments produced similar but more variable results, with amplitudes differing between them more than implicit times. For local response implicit times, the 95% confidence interval of the difference between instruments was approximately ±1 Z-score (±0.9 ms) in either direction. For local response amplitude, it was approximately ±1.6 Z-scores (±0.3 μV). Conclusions Different amplifiers can yield quite different mfERG P1 implicit times, even with identical band-pass settings. However, the reproducibility of mfERG Z-scores across recording instrumentation is relatively high. Comparison of data across systems and laboratories, necessary for multicenter or longitudinal investigations, is facilitated if raw data are converted into Z-scores based on normative data

A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance

Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes

Thyroid Cancer Imaging In Vivo by Targeting the Anti-Apoptotic Molecule Galectin-3

Background The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3. Methods The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific 99mTc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 ?Ci of 99mTc-labeled mAb to galectin-3 (30 ?g protein/in 100 ?l saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera. Findings Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared. Conclusions These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed

Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway

The ubiquitin–proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated

Automatic Morphological Subtyping Reveals New Roles of Caspases in Mitochondrial Dynamics

Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections

WNT/β-catenin signaling regulates mitochondrial activity to alter the oncogenic potential of melanoma in a PTEN-dependent manner

Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors

The controversy of patellar resurfacing in total knee arthroplasty: Ibisne in medio tutissimus?

Early arthroplasty designs were associated with a high level of anterior knee pain as they failed to cater for the patello-femoral joint. Patellar resurfacing was heralded as the saviour safeguarding patient satisfaction and success but opinion on its necessity has since deeply divided the scientific community and has become synonymous to topics of religion or politics. Opponents of resurfacing contend that the native patella provides better patellar tracking, improved clinical function, and avoids implant-related complications, whilst proponents argue that patients have less pain, are overall more satisfied, and avert the need for secondary resurfacing. The question remains whether complications associated with patellar resurfacing including those arising from future component revision outweigh the somewhat increased incidence of anterior knee pain recorded in unresurfaced patients. The current scientific literature, which is often affected by methodological limitations and observer bias, remains confusing as it provides evidence in support of both sides of the argument, whilst blinded satisfaction studies comparing resurfaced and non-resurfaced knees generally reveal equivalent results. Even national arthroplasty register data show wide variations in the proportion of patellar resurfacing between countries that cannot be explained by cultural differences alone. Advocates who always resurface or never resurface indiscriminately expose the patella to a random choice. Selective resurfacing offers a compromise by providing a decision algorithm based on a propensity for improved clinical success, whilst avoiding potential complications associated with unnecessary resurfacing. Evidence regarding the validity of selection criteria, however, is missing, and the decision when to resurface is often based on intuitive reasoning. Our lack of understanding why, irrespective of pre-operative symptoms and patellar resurfacing, some patients may suffer pain following TKA and others may not have so far stifled our efforts to make the strategy of selective resurfacing succeed. We should hence devote our efforts in defining predictive criteria and indicators that will enable us to reliably identify those individuals who might benefit from a resurfacing procedure. Level of evidence V

Worldwide comparison of survival from childhood leukaemia for 1995–2009, by subtype, age, and sex (CONCORD-2): a population-based study of individual data for 89 828 children from 198 registries in 53 countries

Background Global inequalities in access to health care are reflected in differences in cancer survival. The CONCORD programme was designed to assess worldwide differences and trends in population-based cancer survival. In this population-based study, we aimed to estimate survival inequalities globally for several subtypes of childhood leukaemia. Methods Cancer registries participating in CONCORD were asked to submit tumour registrations for all children aged 0-14 years who were diagnosed with leukaemia between Jan 1, 1995, and Dec 31, 2009, and followed up until Dec 31, 2009. Haematological malignancies were defined by morphology codes in the International Classification of Diseases for Oncology, third revision. We excluded data from registries from which the data were judged to be less reliable, or included only lymphomas, and data from countries in which data for fewer than ten children were available for analysis. We also excluded records because of a missing date of birth, diagnosis, or last known vital status. We estimated 5-year net survival (ie, the probability of surviving at least 5 years after diagnosis, after controlling for deaths from other causes [background mortality]) for children by calendar period of diagnosis (1995-99, 2000-04, and 2005-09), sex, and age at diagnosis (< 1, 1-4, 5-9, and 10-14 years, inclusive) using appropriate life tables. We estimated age-standardised net survival for international comparison of survival trends for precursor-cell acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Findings We analysed data from 89 828 children from 198 registries in 53 countries. During 1995-99, 5-year agestandardised net survival for all lymphoid leukaemias combined ranged from 10.6% (95% CI 3.1-18.2) in the Chinese registries to 86.8% (81.6-92.0) in Austria. International differences in 5-year survival for childhood leukaemia were still large as recently as 2005-09, when age-standardised survival for lymphoid leukaemias ranged from 52.4% (95% CI 42.8-61.9) in Cali, Colombia, to 91.6% (89.5-93.6) in the German registries, and for AML ranged from 33.3% (18.9-47.7) in Bulgaria to 78.2% (72.0-84.3) in German registries. Survival from precursor-cell ALL was very close to that of all lymphoid leukaemias combined, with similar variation. In most countries, survival from AML improved more than survival from ALL between 2000-04 and 2005-09. Survival for each type of leukaemia varied markedly with age: survival was highest for children aged 1-4 and 5-9 years, and lowest for infants (younger than 1 year). There was no systematic difference in survival between boys and girls. Interpretation Global inequalities in survival from childhood leukaemia have narrowed with time but remain very wide for both ALL and AML. These results provide useful information for health policy makers on the effectiveness of health-care systems and for cancer policy makers to reduce inequalities in childhood survival