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This study examines “test-wiseness” rules-of-thumb accounting students may use when they cannot answer a multiple-choice question. The effectiveness of rules (whether students comprehend and exploit them) is poorly understood, but they rely largely on flaws in question design. After identifying 11 relevant rules, we first utilize graduate research assistants in an experiment to test the rules' efficacy. Participants successfully use three rules to answer questions on unfamiliar material and are able to discriminate as to the rules' usefulness. To assess general familiarity with and belief in the rules, we survey undergraduate accounting majors at two universities. They demonstrate well-formed ideas of the relative usefulness, which are strongly correlated between universities and moderately correlated with the experimental participants' beliefs. The results illuminate issues that question writers should consider, to avoid vulnerability to test-wiseness or even turn the rules to their advantage when composing questions.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha 1 antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF).Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway.The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild type (WT) A1AT with a 13-fold and 4-fold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the μs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients’ respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a 'stable-labile' design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation. By incorporating thioesters into these bridges, we demonstrate that they are primed for native chemical ligation (NCL) with N-terminal cysteines; offering an alternative to the requirement for C-terminal thioesters for use in such ligations. Alternatively, the use of hydrazine as the ligating nucleophile enables a separate cargo to be attached to each cysteine residue, which are exploited to insert variably cleavable linkers. These methodologies are demonstrated on an antibody fragment, and serve to expand the scope of disulfide bridging strategies whilst offering a convenient route to the construction of multifunctional antibody fragment conjugates.

International Accounting Accreditation Standard A5, issued by the Association to Advance Collegiate Schools of Business (AACSB), requires that information technology competencies be integrated into accounting program curricula. Lee et al. (2018) describe specific software tools used in the accounting profession. As expected, accounting professionals rated Microsoft Excel as the most frequently used software tool. Surprisingly, these professionals rated Adobe Acrobat as the second most frequently used software tool. In a follow-up survey of 286 accounting professionals unique from the respondents in Lee et al. (2018), respondents confirmed the importance of Adobe Acrobat knowledge for accounting graduates entering the profession. This case introduces accounting students to the Adobe Acrobat functions rated most useful to accountants. Students using the case significantly increased their knowledge of the 10 Adobe Acrobat functions accounting professionals rated most important.

Genetic mutations predispose the serine protease inhibitor α1-antitrypsin to misfolding and polymerisation within hepatocytes, causing liver disease and chronic obstructive pulmonary disease. This misfolding occurs via a transiently populated intermediate state, but our structural understanding of this process is limited by the instability of recombinant α1-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α1-antitrypsin at natural isotopic abundance to investigate the consequences of disease-causing mutations, and observe widespread chemical shift perturbations for methyl groups in Z AAT (E342K). By comparison with perturbations induced by binding of a small-molecule inhibitor of misfolding we conclude that they arise from rapid exchange between the native conformation and a well-populated intermediate state. The observation that this intermediate is stabilised by inhibitor binding suggests a paradoxical approach to the targeted treatment of protein misfolding disorders, wherein the stabilisation of disease-associated states provides selectivity while inhibiting further transitions along misfolding pathways.

SUMMARY This study examines the PCAOB's Division of Enforcement from 2005 to 2017, a time period when it expanded from a $5 million to a $22 million program area. We find that a pronounced increase in disciplinary orders issued during the latter years of the sample period is attributable to serious audit deficiencies and misconduct by triennially inspected audit firms, non-U.S. audit firms, and firms auditing brokers and dealers. We also find that more than two-thirds of the violations of PCAOB auditing standards described in these disciplinary orders pertain to failures in general audit principles and responsibilities, obtaining audit evidence, and review and communication. Finally, we find that the PCAOB levies punitive sanctions on an overwhelming majority of audit personnel and audit firms cited in these disciplinary orders. Overall, our results indicate that the PCAOB's enforcement function has actively disciplined audit personnel and audit firms that breached their professional obligations. Data Availability: Publicly available.

α1-Antitrypsin is a protease inhibitor belonging to the serpin family. Serpin polymerisation is at the core of a class of genetic conformational diseases called serpinopathies. These polymers are known to be unbranched, flexible, and heterogeneous in size with a beads-on-a-string appearance viewed by negative stain electron microscopy. Here, we use atomic force microscopy and time-lapse dynamic light scattering to measure polymer size and shape for wild-type (M) and Glu342→Lys (Z) α1-antitrypsin, the most common variant that leads to severe pathological deficiency. Our data for small polymers deposited onto mica and in solution reveal a power law relation between the polymer size, namely the end-to-end distance or the hydrodynamic radius, and the polymer mass, proportional to the contour length. We use the scaling concepts of polymer physics to assess that α1-antitrypsin polymers are random linear chains with a low persistence length.

Severe α1‐antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1‐antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA‐encoded chemical library to undertake a high‐throughput screen to identify small molecules that bind to, and stabilise Z α1‐antitrypsin. The lead compound blocks Z α1‐antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1‐antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1‐antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that “mutation ameliorating” small molecules can block the aberrant polymerisation that underlies Z α1‐antitrypsin deficiency., A chemistry campaign has developed a small molecule that stabilises the severe Z deficiency mutant of α1‐antitrypsin. The lead compound binds to a cryptic pocket and blocks the conformational change and pathological polymerisation that underlie α1‐antitrypsin deficiency.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α

Binge ethanol drinking is an increasingly problematic component of alcohol use disorder costing the United States approximately over $150 billion every year and causes progressive neuroplasticity alterations in numerous brain regions. However, the precise nature or machinery that underlies binge drinking has not yet been elucidated. Corticotropin releasing factor (CRF) neurons in the central amygdala (CeA) are thought to modulate binge drinking, but the specific circuit mechanisms remain poorly understood. Here, we combined optogenetics with in vivo electrophysiology to identify and record from CeA CRF neurons in mice during a repeated binge ethanol drinking task. First, we found that CeA CRF neurons were more active than CeA non-CRF cells during our binge drinking paradigm. We also observed that CeA CRF neurons displayed a heterogeneous spectrum of responses to a lick of ethanol including, pre-lick activated, lick-excited, lick-inhibited, and no response. Interestingly, pre-lick activated CeA CRF neurons exhibited higher frequency and burst firing during binge drinking sessions. Moreover, their overall tonic and phasic electrical activity enhances over repeated binge drinking sessions. Remarkably, CeA CRF units and pre-lick activated CeA CRF neurons did not show higher firing rate or bursting activity during water and sucrose consumption, suggesting that ethanol may “hijack” or plastically alter their intrinsic excitability. This article is part of the special issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse’.

The formation of ordered Z α1‐antitrypsin polymers is central to liver disease in α1‐antitrypsin deficiency. The nascent α1‐antitrypsin folds via the M* intermediate to native monomer or becomes incorporated into a soluble polymer that can be secreted, degraded or precipitate as insoluble inclusions. We describe the kinetics of the clearance of Z α1‐antitrypsin polymers and show that they can be abolished with a small molecule preventing the formation of M*., The formation of ordered Z (Glu342Lys) α1‐antitrypsin polymers in hepatocytes is central to liver disease in α1‐antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation‐selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse‐chase experiments demonstrate that Z α1‐antitrypsin accumulates as short‐chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half‐life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by‐product thereof) was identified in the cells by a conformation‐specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1‐antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.

Severe α1-antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1-antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high throughput screen to identify small molecules that bind to, and stabilise Z α1-antitrypsin. The lead compound blocks Z α1-antitrypsin polymerisationin vitro, reduces intracellular polymerisation and increases the secretion of Z α1-antitrypsin three-fold in mammalian cells including an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerization pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1-antitrypsin into the plasma by 7-fold. There was no observable clearance of hepatic inclusions with respect to controls. This study provides proof-of-principle that ‘mutation ameliorating’ small molecules are a viable approach to treat protein conformational diseases.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%–5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo. To this end, we developed a monoclonal antibody (mAb2H2) that specifically binds to M in preference to Z A1AT, localized its epitope using crystallography to a region perturbed by the Z (Glu342Lys) substitution, and used Fab fragments to label polymers isolated from an MZ heterozygote liver explant. Glu342 is critical to the affinity of mAb2H2, since it also recognized the mild S-deficiency variant (Glu264Val) present in circulating polymers from SZ heterozygotes. Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample. These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals., A monoclonal antibody developed as a single-molecule probe has revealed pathological aggregates of α-1-antitrypsin capture the WT variant.

Science advances 6(43), eabc1370 (1-14) (2020). doi:10.1126/sciadv.abc1370, The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α$_1$-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α$_1$-antitrypsin as “polymer” chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α$_1$-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α$_1$-antitrypsin., Published by Assoc., Washington, DC [u.a.]

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.

Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants. Here we report the identification and characterisation of the novel AAT reactive centre loop variant Gly349Arg (p.G373R) present in the ExAC database. This AAT variant is secreted at normal levels in cellular models of AATD but shows a severe reduction in anti-HNE activity. Biochemical and molecular dynamics studies suggest it exhibits unfavourable RCL presentation to cognate proteases and compromised insertion of the RCL into β-sheet A. Identification of a fully dysfunctional AAT mutant that does not show a secretory defect underlines the importance of accurate genotyping of patients with pulmonary AATD manifestations regardless of the presence of normal levels of AAT in the circulation. This subtype of disease is reminiscent of dysfunctional phenotypes in antithrombin and C1-inibitor deficiencies so, accordingly, we classify this variant as the first pure functionally-deficient (type II) AATD mutant.

Serpin polymerization is the result of end-to-end ordered aggregation of serpin monomers into linear unbranched chains. This change in molecular state represents the basis of several conformational diseases with pathological gain-of-function and loss-of-function phenotypes, termed serpinopathies. Tools that enable quantification and characterization of polymer formation are therefore important to the study of serpin behavior in this pathophysiological context. Such methods rely on different manifestations of molecular change: polymerization-the generation of molecules with increasing molecular weight-is accompanied by concomitant structural rearrangements in the constituent subunits. Different approaches may be appropriate dependent on whether measurements are made on static samples, such as tissue or cell culture extracts, or in time-resolved experiments, often undertaken using polymers artificially induced under in vitro destabilizing conditions. In the former category, we describe the application of polyacrylamide electrophoresis, Western blot, ELISA, and negative-stain electron microscopy and in the latter category, Förster resonance energy transfer and fluorescence spectroscopy using environment-sensitive probes.

Serpin polymerization is the result of end-to-end ordered aggregation of serpin monomers into linear unbranched chains. This change in molecular state represents the basis of several conformational diseases with pathological gain-of-function and loss-of-function phenotypes, termed serpinopathies. Tools that enable quantification and characterization of polymer formation are therefore important to the study of serpin behavior in this pathophysiological context. Such methods rely on different manifestations of molecular change: polymerization-the generation of molecules with increasing molecular weight-is accompanied by concomitant structural rearrangements in the constituent subunits. Different approaches may be appropriate dependent on whether measurements are made on static samples, such as tissue or cell culture extracts, or in time-resolved experiments, often undertaken using polymers artificially induced under in vitro destabilizing conditions. In the former category, we describe the application of polyacrylamide electrophoresis, Western blot, ELISA, and negative-stain electron microscopy and in the latter category, Forster resonance energy transfer and fluorescence spectroscopy using environment-sensitive probes.

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Förster resonance energy transfer (FRET) and gel-based approaches for their characterization.

Severe alpha-1-antitrypsin deficiency (AATD) is most frequently associated with the alpha-1-antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early-onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis (PAGE) and ELISA-based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT.

Many serpinopathies, including alpha-1 antitrypsin (A1AT) deficiency, are associated with the formation of unbranched polymer chains of mutant serpins. In vivo, this deficiency is the result of mutations that cause kinetic or thermodynamic destabilization of the molecule. However, polymerization can also be induced in vitro from mutant or wild-type serpins under destabilizing conditions. The characteristics of the resulting polymers are dependent upon induction conditions. Due to their relationship to disease, serpin polymers, mainly those formed from A1AT, have been widely studied. Here, we describe Forster resonance energy transfer (FRET) and gel-based approaches for their characterization.

As with all proteins, the structural behavior of α 1 -antitrypsin determines its function. However, the functional mechanism by which α 1 -antitrypsin inhibits its target proteases is unusually dynamic, relating to its folding and the potential for it to adopt more thermodynamically stable forms than its native conformation. Alpha1-antitrypsin deficiency arises most commonly as a result of point mutations that affect the structure and dynamic behavior of α 1 -antitrypsin in solution during and after folding. Understanding the dynamic structural behavior of α 1 -antitrypsin, therefore, explains both how it functions in health and how it drives disease. Consequently, it identifies novel therapeutic strategies to treat the underlying pathogenic mechanisms in α 1 -antitrypsin deficiency.

Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate—and therefore polymerize—more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the “breach” region and “shutter” region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

SUMMARY We summarize our recently published study in Accounting Horizons (Catanach et al. 2011) that examines client acceptance patterns and client outcomes following auditor resignations. We used a sample of auditor resignations to examine two issues: (1) why accounting firms assume the role of successor auditor on these presumably risky engagements, and (2) the future outcomes of clients accepted by these successor auditors. We find that smaller accounting firms accept the successor auditor role for resigned clients at a considerably greater rate than do larger firms. Additionally, resigned clients accepted by smaller firms are riskier on several dimensions than those accepted by larger firms. Furthermore, resigned clients accepted by smaller firms are associated with weaker long-term financial ratios, shorter survival tenures, and a greater likelihood of adverse outcomes relative to those accepted by larger firms. We offer related insights for practitioners.

The article provides a full codicological and palaeographical description of a fragment of an evangelistary currently preserved in the composite manuscript Biblioteca Apostolica Vaticana, Vat. lat. 10644, ff. 28r-31r. It identifies the fragment for the first time as a product of the scriptorium of the Abbey of Montecassino at the height of its cultural and ecclesio-political power in the late 11th century. The identification has important implications for earlier identification of other gospel books produced at the Abbey which are discussed here.

SYNOPSIS Under the fair value option, SFAS No. 159, firms have full discretion over electing to report specified financial instruments at fair value on a contract-by-contract basis. Building on Henry's (2009) study of early adopting banks, this paper examines to what extent firms' election of instruments benefited their current or future earnings. Our sample comprises the constituents of the S&P 1500 Index for the first quarters of fiscal years 2007 and 2008. Expanding the sample across industries and over time allows us to obtain a more complete picture of the adoption of the fair value option. We identify 72 adopters, two-thirds of which are not commercial banks. We do not find evidence of systematic opportunistic election of the fair value option. In only a handful of cases—concentrated among early adopters with an earnings shortfall—did firms experience a significant improvement in current or future earnings that casts doubt on whether their adoption was keeping with the intent and spirit of the standard. Data Availability: The list of adopters used in this paper is available from the authors upon request.

Members of the serine protease inhibitor (serpin) superfamily are found in all branches of life and play an important role in the regulation of enzymes involved in proteolytic cascades. Mutants of the serpins result in a delay in folding, with unstable intermediates being cleared by endoplasmic reticulum-associated degradation. The remaining protein is either fully folded and secreted or retained as ordered polymers within the endoplasmic reticulum of the cell of synthesis. This results in a group of diseases termed the serpinopathies, which are typified by mutations of a1-antitrypsin and neuroserpin in association with cirrhosis and the dementia familial encephalopathy with neuroserpin inclusion bodies, respectively. Current evidence strongly suggests that polymers of mutants of a1-antitrypsin and neuroserpin are linked by the sequential insertion of the reactive loop of one molecule into b-sheet A of another. The ordered structure of the polymers within the endoplasmic reticulum stimulates nuclear factor-kappa B by a pathway that is independent of the unfolded protein response. This chronic activation of nuclear factor-kappa B may contribute to the cell toxicity associated with mutations of the serpins. We review the pathobiology of the serpinopathies and the development of novel therapeutic strategies for treating the inclusions that cause disease. These include the use of small molecules to block polymerization, stimulation of autophagy to clear inclusions and stem cell technology to correct the underlying molecular defect.

SYNOPSIS The auditor change literature has generally concluded that clients from whom an audit firm resigns are risky clients, yet little is known about the period after a predecessor auditor has resigned from an engagement. We investigate a sample of resignations to determine why an audit firm chooses to accept the role of successor auditor on a presumably risky engagement and whether this decision is associated with a future adverse outcome. Consistent with prior studies, our results indicate that, relative to Non-Big N firms, Big N firms are more selective in accepting the successor auditor role when the predecessor auditor has resigned. Incremental to these prior studies, we find that Big N firms factor in two variables to help mitigate their potential risk—the timing of the predecessor audit firm's resignation and their own firm's expertise. Our analysis of future outcomes indicates that the resigned clients engaged by Non-Big N successor auditors are associated with weaker long-term financial ratios, shorter survival tenures, and a greater proportion of adverse outcomes compared with the resigned clients engaged by Big N successor auditors. Data Availability: Data are available from the sources indicated in the text.

In this study, we document a significant shift over the past several years from stock option-based compensation to restricted stock-based compensation. Additionally, we evaluate whether stock option grants and restricted stock grants result in similar valuation consequences for firms. We estimate cross-sectional valuation equations that include the value of stock option and restricted stock grants summed over the current and past two years, residual income, and book value of equity, after controlling for endogeneity. Consistent with prior research, our findings indicate that the market on average values stock option grants positively. However, in contrast to stock option grants, restricted stock grants are valued negatively. This result is consistent with restricted stock grants lacking the positive incentive effects of stock options and being viewed as a liability or expense to the firm.

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.

The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 A and 2.0 A resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical "canonical" fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

A gene encoding a novel component of the cellulolytic complex (cellulosome) of the anaerobic fungus Piromyces sp. strain E2 was identified. The encoded 538 amino acid protein, named celpin, consists of a signal peptide, a positively charged domain of unknown function followed by two fungal dockerins, typical for components of the extracellular fungal cellulosome. The C-terminal end consists of a 380 amino acid serine proteinase inhibitor (or serpin) domain homologue, sharing 30 % identity and 50 % similarity to vertebrate and bacterial serpins. Detailed protein sequence analysis of the serpin domain revealed that it contained all features of a functional serpin. It possesses the conserved amino acids present in more than 70 % of known serpins, and it contained the consensus of inhibiting serpins. Because of the confined space of the fungal cellulosome inside plant tissue and the auto-proteolysis of plant material in the rumen, the fungal serpin is presumably involved in protection of the cellulosome against plant proteinases. The celpin protein of Piromyces sp. strain E2 is the first non-structural, non-hydrolytic fungal cellulosome component. Furthermore, the celpin protein of Piromyces sp. strain E2 is the first representative of a serine proteinase inhibitor of the fungal kingdom.

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa alpha-helix 163-170 (h163-170), Arg(165) and Lys(169), participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH(2)-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na(+) because using a higher Na(+) concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH(2)-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na(+)-binding site of the protease and that residues Val(163) and Ser(167) play a key role in this interaction.

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.

CCR5 is a chemokine receptor that mediates entry of human immunodeficiency virus-1 (HIV-1). Two monoclonal antibodies (mAbs) that block HIV-1 entry, 3A9 and 5C7, were used to select peptide mimotopes of sequences on CCR5 from phage displayed peptide libraries. The selected mimotofpes comprised motifs at the N-terminus and on the first and third extracellular loops (ECL1 and ECL3) of CCR5. Amino acids in these motifs were exchanged for alanines by site-directed mutagenesis (sdm) in the cDNA for human CCR5. Ensuing effects on antibody binding to CCR5, cellular entry of HIV-1 and chemokine-induced signalling were analysed by transfection of mutant cDNAs into HEK293.CD4 cells. For both mAbs, fluorescence-activated cell sorting analysis was used to define overlapping conformational epitopes on CCR5 at the N-terminus, on ECL1 and ECL3. Mutation of the N-terminal motif 10YD11 prevented HIV-1 entry into transfected cells as judged by single round infection assays with R5 and R5X4 HIV-1 isolates, as did mutation of the motif 96FG97 in ECL1, whereas mutation of the motif 274RLD276 in ECL3 had only a minor effect. None of the motifs in CCR5 relevant to HIV-1 entry disrupted chemokine-induced signalling. Thus, peptide mimotopes of conformational contact sites of CCR5 with the paratope of mAbs 3A9 and 5C7 represent sites on CCR5 that are essential for HIV-1 entry. Structural knowledge of these mimotopes could help elucidate the nature of the interaction between CCR5 and HIV-1, and thus the derivation of specific inhibitors of entry of HIV-1 into susceptible cells without interference with chemokine signalling.

A comparison of micropore size distributions in the range below 100 A has been made for a silica gel sample, using the results of exact and approximate Barrett, Joyner & Halenda computations and of the recently proposed method of Dollimore & Heal. A number of discrepancies were noted between the results of the computations; in particular, while total surface areas obtained from the determinations of Barrett, Joyner & Halenda were within 2% of the BET surface area, the value from the Dollimore & Heal computation was about 20% smaller. The various methods which have been proposed for pore size distribution are essentially the same in theory; discrepancies such as those noted in this work are attributed to the differing values of adsorbed multilayer thickness used in the calculations. Although there is little agreement among the published correlations of multilayer thickness for nitrogen adsorption, the Halsey eqauation which is employed by Dollimore & Heal, lies well above other correlations and must be regarded as of questionable value in distribution computations. A satisfactory multilayer thickness correlation should provide a means of accounting for the effects of pore structure and non-ideal adsorption; the approach suggested by Mingle & Smith may be promising in this direction.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.