Targeted Mass Spectrometry-Based Approach for the Determination of Intrinsic Internalization Kinetics of Cell-Surface Membrane Protein Targets.

Successful development of targeted therapeutics aimed at the elimination of diseased cells relies on the target properties and the therapeutics that target them. Currently, target properties have been evaluated through antibody-dependent semiquantitative approaches such as flow cytometry, Western blotting, or microscopy. Since antibodies can alter target properties following binding, antibody-dependent approaches provide at best skewed measurements for target intrinsic properties. To circumvent, here we attempted to develop an antibody-free targeted mass spectrometry-based (ATM) strategy to measure the surface densities and the intrinsic rates (Kint) of CD38 internalization in multiple myeloma cell lines. Using cell-surface biotinylation in conjunction with differential mass tagging to separate inward CD38 molecules from the outbound and nascent ones, the ATM approach revealed diversities in measured CD38 Kint values of 0.239 min-1 S.E. ± 0.076, 0.109 min-1 S.E. ± 0.032, and 0.058 min-1 S.E. ± 0.001 for LP1, NCIH929, and MOLP8 cell lines, respectively. Together with CD38 surface densities, intrinsic Kint values aligned well with the tumor penetration model and supported the outcomes for tumor regression in mouse xenografts upon drug treatment. Additionally, the ATM approach can evaluate molecules with fast Kint as we determined for CTLA4 protein. We believe that the ATM approach has the potential to evaluate diverse cell-surface targets as part of the pharmacological assessment in drug discovery.

Imaging of small animal peripheral artery disease models: recent advancements and translational potential.

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic.

Diagnosis and treatment of gallstone disease.

Gallstones form when there is an imbalance in the composition of bile resulting in precipitation of one or more of its components. Between 37 and 86% of gallstones are cholesterol-rich stones, 2-27% are pigment stones and 4-16% are mixed. Cholesterol-rich gallstones are more common in Europe and North America. This has been attributed to obesity and diets containing a high proportion of refined carbohydrates and fat. Low-calorie diets and rapid weight loss are also associated with cholesterol-rich gallstones. Gallstone disease increases with age. Women have a higher prevalence of gallstones than men, which is attributed to exposure to oestrogen and progesterone. Of those with gallstones, around 1 to 4% will develop symptoms annually. Most patients (> 80%) will remain asymptomatic throughout their lifetime and the likelihood of developing symptoms diminishes with time. Liver function tests and an abdominal ultrasound should be offered to patients with symptoms suggestive of gallstone disease (e.g. abdominal pain, jaundice, fever). They should also be considered in patients with less typical but chronic abdominal or gastrointestinal symptoms. In patients with acute pancreatitis and evidence of ongoing bile duct obstruction and/or cholangitis, endoscopic retrograde cholangio-pancreatography and biliary sphincterotomy is recommended within 24-72 hours of the onset of symptoms. Patients with acute cholecystitis should be referred for laparoscopic cholecystectomy.

Broad proteomics analysis of seeding-induced aggregation of α-synuclein in M83 neurons reveals remodeling of proteostasis mechanisms that might contribute to Parkinson's disease pathogenesis.

Aggregation of misfolded α-synuclein (α-syn) is a key characteristic feature of Parkinson's disease (PD) and related synucleinopathies. The nature of these aggregates and their contribution to cellular dysfunction is still not clearly elucidated. We employed mass spectrometry-based total and phospho-proteomics to characterize the underlying molecular and biological changes due to α-syn aggregation using the M83 mouse primary neuronal model of PD. We identified gross changes in the proteome that coincided with the formation of large Lewy body-like α-syn aggregates in these neurons. We used protein-protein interaction (PPI)-based network analysis to identify key protein clusters modulating specific biological pathways that may be dysregulated and identified several mechanisms that regulate protein homeostasis (proteostasis). The observed changes in the proteome may include both homeostatic compensation and dysregulation due to α-syn aggregation and a greater understanding of both processes and their role in α-syn-related proteostasis may lead to improved therapeutic options for patients with PD and related disorders.

Active pulmonary tuberculosis: something old, something new, something borrowed, something blue.

Tuberculosis remains a major global health issue affecting all countries and age groups. Radiology plays a crucial role in the diagnosis and management of pulmonary tuberculosis (PTB). This review aims to improve understanding and diagnostic value of imaging in PTB. We present the old, well-established findings ranging from primary TB to the common appearances of post-primary TB, including dissemination with tree-in-bud nodularity, haematogenous dissemination with miliary nodules and lymphatic dissemination. We discuss new concepts in active PTB with special focus on imaging findings in immunocompromised individuals. We illustrate PTB appearances borrowed from other diseases in which the signs were initially described: the reversed halo sign, the galaxy sign and the cluster sign. There are several radiological signs that have been shown to correlate with positive or negative sputum smears, and radiologists should be aware of these signs as they play an important role in guiding the need for isolation and empirical anti-tuberculous therapy.

Control of centromere localization of the MEI-S332 cohesion protection protein.

In mitosis and meiosis, cohesion is maintained at the centromere until sister-chromatid separation. Drosophila MEI-S332 is essential for centromeric cohesion in meiosis and contributes to, though is not absolutely required for, cohesion in mitosis. It localizes specifically to centromeres in prometaphase and delocalizes at the metaphase-anaphase transition. In mei-S332 mutants, centromeric sister-chromatid cohesion is lost at anaphase I, giving meiosis II missegregation. MEI-S332 is the founding member of a family of proteins important for chromosome segregation. One likely activity of these proteins is to protect the cohesin subunit Rec8 from cleavage at the metaphase I-anaphase I transition. Although the family members do not show high sequence identity, there are two short stretches of homology, and mutations in conserved residues affect protein function. Here we analyze the cis- and trans-acting factors required for MEI-S332 localization. We find a striking correlation between domains necessary for MEI-S332 centromere localization and conserved regions within the protein family. Drosophila MEI-S332 expressed in human cells localizes to mitotic centromeres, further highlighting this functional conservation. MEI-S332 can localize independently of cohesin, assembling even onto unreplicated chromatids. However, the separase pathway that regulates cohesin dissociation is needed for MEI-S332 delocalization at anaphase.

The meaning of excellence.

RATIONALE AND OBJECTIVES: Program directors would like to interview the very best students applying to their programs. The summary paragraph of the dean's letter should provide useful information regarding a student's performance in medical school. One frequently found descriptor is excellent. However, its very frequency suggests the word may be loosely used. The purpose of this investigation is to determine the meaning of excellence. MATERIALS AND METHODS: The descriptor excellent was searched for in the summary paragraph. An effort was made to determine how many medical schools used excellent, how precisely the medical school defined this word, whether numbers were used to define the upper and lower boundaries of excellent, and what other buzzwords were used in the summary paragraphs for students not defined as excellent. RESULTS: Excellent was the most common descriptor, used by 75% of the medical schools. Defined numeric boundaries were used by 47% of schools. Tabulated results showed that within a school the range of excellence varied from as tight as 20 percentile points to so broad that 65% of the students were classified as excellent. The boundaries of excellent, among different schools, varied from as low as the third to as high as the ninety-second percentile. In half the schools, students described as excellent might be in the bottom half of their class. A total of 28% of the schools used excellent, but without any numeric definition. No school used excellent to describe its best students. CONCLUSIONS: Medical student deans often exaggerate the quality of their graduates by using the word excellent at variance with the dictionary definition of exceptionally good. Inaccurate descriptions by deans of their graduating medical students diminish the value of MSPE.

The cephalic phase insulin response to nutritive and low-calorie sweeteners in solid and beverage form.

The purpose of the study was to examine the role of the cephalic phase insulin response (CPIR) following exposure to nutritive and low-calorie sweeteners in solid and beverage form in overweight and obese adults. In addition, the role of learning on the CPIR to nutritive and low-calorie sweetener exposure was tested. Sixty-four overweight and obese adults (age: 18-50years, BMI: 24-37kg/m2, body fat percentage>25% for men and >32% for women) were sham-fed (at 2-minute intervals for 14min) a randomly assigned test load comprised of a nutritive (sucrose) or low-calorie sweetener (sucralose) in beverage or solid form in phase 1 of the study. A 2-3ml blood sample was collected before and 2, 6, 10, 14, 61, 91 and 121min after oral exposure for serum insulin and glucose analysis. During phase 2, participants underwent a 2-week training period to facilitate associative learning between the sensory properties of test loads and their post-ingestive effects. In phase 3, participants were retested for their cephalic phase responses as in phase 1. Participants were classified as responders if they demonstrated a positive insulin response (rise of serum insulin above baseline i.e. Δ insulin) 2min post-stimulus in phase 1. Among responders exposed to the same sweetener in Phases 1 and 3, the proportion of participants that displayed a rise of insulin with oral exposure to sucralose was significantly greater when the stimulus was in the solid form compared to the beverage form. Sucralose and sucrose exposure elicited similarly significant increases in serum insulin 2min after exposure and significant decreases after 2min in responders in both food forms. The solid food form elicited greater CPIR over 2, 6 and 10min than the beverage form. There was no effect of learning on insulin responses after training. The results indicate the presence of a significant CPIR in a subset of individuals with overweight or obesity after oral exposure to sucralose, especially when present in solid food form. Future studies must confirm the reliability of this response.

The E2F cell cycle regulator is required for Drosophila nurse cell DNA replication and apoptosis.

During Drosophila oogenesis nurse cells become polyploid, enabling them to provide the developing oocyte with vast amounts of maternal messages and products. The nurse cells then die by apoptosis. In nurse cells, as in many other polyploid or polytene tissues, replication is differentially controlled and the heterochromatin is underreplicated. The nurse cell chromosomes also undergo developmentally induced morphological changes from being polytene, with tightly associated sister chromatids, to polyploid, with dispersed sister chromatids. We used female-sterile dE2F1 and dDP mutants to assess the role of the E2F cell cycle regulator in oogenesis and the relative contributions of transcriptional activation versus repression during nurse cell development. We report here that E2F1 transcriptional activity in nurse cells is essential for the robust synthesis of S-phase transcripts that are deposited into the oocyte. dE2F1 and dDP are needed to limit the replication of heterochromatin in nurse cells. In dE2F1 mutants the nurse cell chromosomes do not properly undergo the transition from polyteny to polyploidy. We also find that dDP and dE2F1 are needed for nurse cell apoptosis, implicating transcriptional activation of E2F target genes in this process.

Identification of a lung cancer cell line deficient in atg7-dependent autophagy.

Autophagy is a major cellular process for bulk degradation of proteins and organelles in order to maintain metabolic homeostasis, and it represents an emerging target area for cancer. Initially proposed to be a cancer-restricting process for tumor initiation, recent studies suggest that autophagy can also promote cell survival in established tumors. ATG7 is an essential autophagy gene that encodes the E1 enzyme necessary for the lipidation of the LC3 family of ubiquitin-like proteins and autophagosome formation. In this study we identified a rare case of a cancer cell line, H1650 lung adenocarcinoma, which has lost ATG7 expression due to a focal biallelic deletion within the ATG7 locus. These cells displayed no evidence of ATG7 pathway activity; however, reconstituting the cells with wild-type ATG7 restored both LC3 lipidation and downstream autophagic consumption of autophagy substrates such as the SQSTM1/p62 protein. We characterized several phenotypes reported to be influenced by autophagy, and observed an ATG7-dependent increase in cell growth and clearance of proteasome-inhibitor induced protein aggregates. Cellular changes in mitochondrial metabolism or response to nutrient starvation were unaffected by ATG7 expression. In addition, parental H1650 cells that lacked ATG7 were still able to consume autophagy substrates SQSTM1, NBR1 and TAX1BP1 via a bafilomycin A1-sensitive pathway, suggesting that these proteins were not exclusively degraded by autophagy. Overall, these findings highlight a unique outlier instance of complete loss of ATG7-dependent autophagy in a cancer cell line. The H1650 cell line may be a useful system for future studies to further understand the role of autophagy in tumorigenesis and potential redundant pathways that allow cells to circumvent the loss of ATG7-dependent autophagy in cancer.

Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in autophagosome formation.

Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, we have identified a link between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, we find that paxillin is redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy.

Roles and regulation of the Drosophila centromere cohesion protein MEI-S332 family.

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of