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The article offers information on several court cases related to the violation of securities law in the U.S. including, one between plaintiff Securities and Exchange Commission (SEC) and defendant Rorech, one between appellant Robert Morrison and appellee National Australia Bank Ltd., and one between petitioner Merck & Co. Inc. and respondent Richard Reynolds. The rulings declared by U.S. court in all these cases have also been focused in the article.

The article discusses several court cases concerning the liability of professionals, officers and directors in the U.S. It highlights the "Krier v. Vilione" case which focuses on the duties of an accountant to third parties. It stresses the "Armstrong v. American Pallet Leasing Inc." case which concentrates on the violations of the Racketeer Influenced and Corrupt Organizations Act. It underlines the "Leder v. Shenfeld" case regarding the fiduciary duty between an accountant and a client.

Abstract: Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested. [Copyright &y& Elsevier]

The article reports on the developments in law governing the liability of professionals, officers and directors in the U.S. The past year saw a trend expanding the class of plaintiffs who are entitled to sue an attorney for legal malpractice to include an insurer that asserts a claim against counsel the insurer has assigned to represent its insured. Some courts held that the insured's attorney owes a duty of care not only to the insured, but also to the insurer, whereas other courts allowed the insurer to maintain the action under an equitable subrogation theory. Cases within the past year have focused on the discovery rule and continuous representation rule as doctrines that postpone the accrual of a legal malpractice claim or toll the statute of limitations period for such claims.

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV. [ABSTRACT FROM AUTHOR]

Abstract: Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug–drug interactions with other medicines if developed as therapeutic agents. [Copyright &y& Elsevier]

Caleosin is a Ca2+-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing α- and δ-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid. [ABSTRACT FROM AUTHOR]

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Cav3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Cav3.3-evoked T-type calcium current without altering other biophysical properties of Cav3.3. Immunostaining studies revealed co-localization of CatSper1 and Cav3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Cav3.3 channel, which might be important in the regulation of sperm function. [ABSTRACT FROM AUTHOR]

In plant cells, certain membrane proteins move by unknown mechanisms directly from the endoplasmic reticulum (ER) to prevacuolar or vacuole-like organelles where membrane is internalized to form a dense, lattice-like structure. Here, we identify a sequence motif, PIEPPPHH, in the cytoplasmic tail of a membrane protein that directs the protein from the ER to vacuoles where it is internalized. A type II membrane protein in Arabidopsis thaliana, (At)SRC2 (for Soybean Gene Regulated by Cold-2), binds specifically to PIEPPPHH and moves from the ER to the same vacuoles where it is internalized. Not all proteins that move in this pathway are internalized because another Arabidopsis type II membrane protein, (At)VAP (for Vesicle-Associated Protein), localizes to the same organelles but remains exposed on the limiting membrane. The identification of (At)SRC2 and its preference for interaction with a targeting motif specific for the ER-to-vacuole pathway may provide tools for future dissection of mechanisms involved in this unique trafficking system. [ABSTRACT FROM AUTHOR]

The protein storage vacuole (PSV) is a specialized organelle in plant seeds that accumulates storage proteins and phytate during seed development. In many plant species, such as tomato and tobacco, the PSV contains two types of microscopically visible intra-organellar inclusions: a large crystalline lattice of membranes and proteins, the crystalloid, and one or a few large phytate crystals, the globoids. In seeds of the family Brassicaceae, the PSVs lack visible crystalloids and have many small globoids dispersed throughout. We biochemically fractionated PSVs fromBrassica napusand defined a crystalloid-like fraction that contained integral membrane protein markers found in crystalloids of other plants. Protein analyses identified a previously undescribed family of proteins, the Brassicaceae PSV-embedded proteins (BPEPs), associated with‘crystalloid’ and globoid fractions. The defining characteristics of the BPEPs are an N-terminal signal peptide and tandem MATH domains, which may mediate protein–protein interactions. Database analyses indicated that the BPEPs are unique to Brassicaceae. Immunofluorescence studies using anti-BPEP antibodies and antibodies to other biochemical markers to labelB. napusandArabidopsis thalianaseed sections localized the BPEPs to structures within the PSVs, whose appearance was consistent with a diffuse network of internalized membranes and globoids. These results demonstrate that Brassicaceae PSVs contain internalized membranes, and raise the possibility that BPEPs modify these internal membrane structures to yield a PSV morphology different from that of tomato or tobacco. [ABSTRACT FROM AUTHOR]

Autophagy in plant cells is induced by nutrient starvation. Initially, double membrane-bound organelles, termed autophagosomes, enclose a portion of cytoplasm, and then fuse with a vacuole or lysosome to give an autolysosome. Autolysosomes can be visualized by incubating cells in the presence of a membrane-permeable cysteine protease inhibitor. The inhibitor presumably decreases proteolytic degradation of the autolysosome contents that are composed of portions of cytoplasm enclosed by the membrane originating from the inner membrane of autophagosomes, and allows them to accumulate. The origin of membranes that give rise to autophagosomes and autolysosomes is unknown. Here we use an acidotropic fluorescent dye, LysoTracker Red, to label autolysosomes specifically. We demonstrate that autolysosome membranes are marked by the presence of α-tonoplast intrinsic protein (α-TIP) but not by γ-TIP or δ-TIP. The identification of a TIP specifically associated with membranes derived from an autophagic process may help our understanding of how plant cells generate and maintain functionally distinct types of vacuoles. [ABSTRACT FROM PUBLISHER]

Context Medical educators have emphasized the importance of teaching patient-centred care. Objectives To describe and quantify the attitudes of medical students towards patient-centred care and to examine: (a) the differences in these attitudes between students in early and later years of medical school; and (b) factors associated with patient-centred attitudes. Methods We surveyed 673 students in the first, third, and fourth years of medical school. Our survey utilized the Patient–Practitioner Orientation Scale (PPOS), a validated instrument designed to measure individual preferences towards various aspects of the doctor–patient relationship. Total PPOS scores can range from patient-centred (egalitarian, whole person oriented) to disease- or doctor-centred (paternalistic, less attuned to psychosocial issues). Additional demographic data including gender, age, ethnicity, undergraduate coursework, family medical background and specialty choice were collected from the fourth year class. Results A total of 510 students (76%) completed data collection. Female gender (P < 0·001) and earlier year of medical school (P = 0·03) were significantly associated with patient-centred attitudes. Among fourth year students (n = 89), characteristics associated with more patient-centred attitudes included female gender, European-American ethnicity, and primary-care career choice (P < 0·05 for each comparison). Conclusion Despite emphasis on the need for curricula that foster patient-centred attitudes among medical students, our data suggest that students in later years of medical school have attitudes that are more doctor-centred or paternalistic compared to students in earlier years. Given the emphasis placed on patient satisfaction and patient-centred care in the current medical environment, our results warrant further research and dialogue to explore the dynamics in medical education that may foster or inhibit student attitudes toward... [ABSTRACT FROM AUTHOR]

Examines the deposits of storage proteins into protein storage vacuoles (PSV) during plant seed development and maturation. Role of storage proteins during germination; Presence of organelles within the secretory pathway of eukaryotic cells; Examination on the nature of globoid membrane.

The development of the TMTpro-16plex series expanded the breadth of commercial isobaric tagging reagents by nearly 50% over classic TMT-11plex. In addition to the described 16plex reagents, the proline-based TMTpro molecule can accommodate two additional combinations of heavy carbon and nitrogen isotopes. Here, we introduce the final two labeling reagents, TMTpro-134C and TMTpro-135N, which permit the simultaneous global protein profiling of 18 samples with essentially no missing values. For example, six conditions with three biological replicates can now be perfectly accommodated. We showcase the 18plex reagent set by profiling the proteome and phosphoproteome of a pair of isogenic mammary epithelial cell lines under three conditions in triplicate. We compare the depth and quantitative performance of this data set with a TMTpro-16plex experiment in which two samples were omitted. Our analysis revealed similar numbers of quantified peptides and proteins, with high quantitative correlation. We interrogated further the TMTpro-18plex data set by highlighting changes in protein abundance profiles under different conditions in the isogenic cell lines. We conclude that TMTpro-18plex further expands the sample multiplexing landscape, allowing for complex and innovative experimental designs.

Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+. We label each peptide with TMTproZero or Super Heavy TMTpro reagents and separately spike each peptide into a TMTpro16-labeled background (equal amount of peptide across all 16 channels). We observe that the expected 1:1 reporter ion ratio is distorted when a TMTproZero-labeled peptide is used; however, we note no such interference when shTMTpro substitutes the TMTproZero tag. Our data suggest that using the Super Heavy TMTpro reagent is an improvement over the TMTproZero reagent for the accurate quantification of high-charge-state peptides for trigger-based multiplexed assays.

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.

Mass spectrometry (MS) has become the predominant technology to analyze proteins due to it ability to identify and characterize proteins and their modifications with high sensitivity and selectivity (Aebersold and Mann, Nature 422(6928):198-207, 2003; Han et al., Curr Opin Chem Biol 12(5):483-490, 2008). While mass spectrometry instruments have improved rapidly over the past couple of decades, mass spectrometry results have remained largely dependent on sample preparation and quality. Sample ionization and mass measurements are susceptible to a wide variety of interferences, including buffers, salts, polymers, and detergents. These contaminants also impair MS system performance, often requiring time consuming maintenance or costly repairs to restore function. The goal of this chapter is to describe the rationale, considerations, and general techniques used to prepare samples for proteomic mass spectrometry analysis.

In receptor-mediated sorting of soluble protein ligands in the endomembrane system of eukaryotic cells, three completely different receptor proteins for mammalian (mannose 6-phosphate receptor), yeast (Vps10p) and plant cells (vacuolar sorting receptor; VSR) have in common the features of pH-dependent ligand binding and receptor recycling. In striking contrast, the plant receptor homology-transmembrane-RING-H2 (RMR) proteins serve as sorting receptors to a separate type of vacuole, the protein storage vacuole, but do not recycle, and their trafficking pathway results in their internalization into the destination vacuole. Even though plant RMR proteins share high sequence similarity with the best-characterized mammalian PA-TM-RING family proteins, these two families of proteins appear to play distinctly different roles in plant and animal cells. Thus, this minireview focuses on this unique sorting mechanism and traffic of RMR proteins via dense vesicles in various plant cell types., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Introduction: This study compared the frequency of oral counseling and written information by primary care physicians at paper medical record (PMR) clinics and electronic medical record (EMR) clinics, and assessed relationships between medication counseling and medication outcomes (knowledge, questions, reported adherence and side effects, and medication fill)., Methods: A cross-sectional study with two convenience samples of English-speaking adult patients receiving > or =1 prescription at the primary care index visit was conducted in two PMR clinics, with 184 (48% response) patients seen by one of 22 physicians, and in two EMR clinics, with 249 (37% response) patients seen by one of 25 physicians. Data were from medical record reviews of the index visit and 2-week post-visit telephone interviews., Results: Three mutually exclusive counseling categories were evaluated. Patients received 1,095 prescriptions, 61% with oral counseling for indications, 21% with oral counseling for indications and side effects, and 12% with written information plus oral ("multi-mode") counseling. General linear mixed models found 1) less multi-mode counseling in PMR clinics (2%) than EMR clinics (20%); 2) PMR and EMR clinics were similar in oral counseling for indications and side effects; and 3) PMR clinics provided more oral counseling only for indications (69%) than EMR (53%) clinics. The impact of receiving oral or written counseling on patients' reports of having questions about their medications was inconclusive. Not receiving oral counseling for indications was associated with more questions, but not receiving written information was associated with fewer questions. Filling a prescription was lower when no oral counseling for indications and side effects was reported, but the absence of written information was associated with more prescriptions fills., Conclusions: Physicians' use of EMR to print medication information did not seem to compromise their oral counseling for medication indications and side effects. This feature of the EMR was underutilized by physicians; however, future studies addressing patient recall and evaluating the quality and content of medication counseling are needed.

Background and Objectives: Previous reports have defined the time that community preceptors spend teaching students, but much remains to be learned about the process and content of office-based teaching. Our objective was to understand better the teaching process and content by documenting how often preceptors used the microskills described in the Five-step "Microskills" Model of Clinical Teaching and how often they discussed tasks described in the Task-oriented Processes in Care (TOPIC) teaching model when working with their students., Methods: Using a checklist combining these two models, two independent observers documented the teaching and learning that occurred between 12 preceptors and their students., Results: Inter-rater concordance was 96.2%. Preceptors frequently used two of the teaching microskills, "probe for supporting evidence" and "teach general rules." Preceptors and students frequently discussed many information-processing and management tasks in the TOPIC model., Conclusions: Despite the lack of training in using either model, preceptors frequently used some teaching microskills and discussed many TOPIC model tasks. This finding supports both models as relevant teaching models but further observational study of preceptors trained in using both models is needed. For the TOPIC model, preceptor development may help preceptors to more explicitly refer to TOPIC tasks when teaching clinical content to students.