Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Altered gene expression patterns of innate and adaptive immunity pathways in transgenic rainbow trout harboring Cecropin P1 transgene.

BACKGROUND: We have recently developed several homozygous families of transgenic rainbow trout harbouring cecropin P1 transgene. These fish exhibit resistance characteristic to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). In our earlier studies we have reported that treatment of a rainbow trout macrophage cell line (RTS11) with a linear cationic α-helical antimicrobial peptide (e.g., cecropin B) resulted in elevated levels of expression of two pro-inflammatory relevant genes (e.g., IL-1ß and COX-2). Therefore, we hypothesized that in addition to the direct antimicrobial activity of cecropin P1 in the disease resistant transgenic rainbow trout, this antimicrobial peptide may also affect the expression of immune relevant genes in the host. To confirm this hypothesis, we launched a study to determine the global gene expression profiles in three immune competent organs of cecropin P1 transgenic rainbow trout by using a 44k salmonid microarray. RESULTS: From the microarray data, a total of 2480 genes in the spleen, 3022 in the kidney, and 2102 in the liver were determined as differentially expressed genes (DEGs) in the cecropin P1 transgenic rainbow trout when compared to the non-transgenics. There were 478 DEGs in common among three tissues. Enrichment analyses conducted by two different bioinformatics tools revealed a tissue specific profile of functional pathway perturbation. Many of them were directly related to innate immune system such as phagocytosis, lysosomal processing, complement activation, antigen processing/presentation, and leukocyte migration. Perturbation of other biological functions that might contribute indirectly to host immunity was also observed. CONCLUSIONS: The gene product of cecropin P1 transgene produced in the disease resistant transgenic rainbow trout not only can kill the pathogens directly but also exert multifaceted immunomodulatory properties to boost host immunity. The identified genes involved in different pathways related to immune function are valuable indicators associated with enhanced host immunity. These genes may serve as markers for selective breeding of rainbow trout or other aquaculture important fish species bearing traits of disease resistance.

Suppliers' Perspectives on Cage-Free Eggs in China.

Successful promotion of cage-free eggs supports a housing system offering potential for improved hen welfare. As the world's largest egg producer and consumer, China offers much potential for welfare improvements. We examined 10 Chinese companies supplying cage-free eggs (four using indoor systems, six with outdoor access) to understand their strategies to promote cage-free eggs to businesses and consumers. We purposively sampled 12 employees from these companies familiar with production or sales. We conducted two-three semi-structured interviews per participant, collected public online documents (including online shops and social media content), and recorded field notes. We analyzed the data using template analysis to generate key results. Participants reported buyers being unfamiliar with 'animal welfare' and 'cage-free', but familiar with concepts associated with 'free-range'. Participants considered three attributes when promoting cage-free eggs: price (engaging buyers who were willing to pay more), experiential attributes (e.g., taste, accommodating buyer preferences), and non-sensory credence attributes (e.g., cage-free production, improving buyers' understanding and trust). Our results are not generalizable, though they may be transferable to similar contexts. Understanding how companies promoted cage-free eggs to buyers may help inform promotion of other animal products with welfare attributes. Simultaneous efforts are needed to ensure actual welfare improvements on farms.

Safety of Deutetrabenazine for the Treatment of Tardive Dyskinesia and Chorea Associated with Huntington Disease.

INTRODUCTION: Deutetrabenazine is a vesicular monoamine transporter 2 inhibitor used to treat tardive dyskinesia (TD) and chorea associated with Huntington disease (HD). To enhance detection of safety signals across individual trials, integrated safety analyses of deutetrabenazine in TD and HD chorea were conducted. METHODS: For TD, safety data were integrated from two 12-week pivotal studies (ARM-TD and AIM-TD) and through week 15 of the open-label extension (OLE) study (RIM-TD). Data were analyzed by deutetrabenazine treatment group and placebo. For HD, safety data were integrated from the 12-week pivotal study (First-HD) and through week 15 of the OLE study (ARC-HD) for patients previously receiving placebo. Integrated deutetrabenazine data were compared with placebo from the pivotal study. RESULTS: For TD, deutetrabenazine (n = 384) was generally well tolerated compared with placebo (n = 130). Adverse event (AE) incidence was numerically higher in the response-driven deutetrabenazine vs the fixed-dose deutetrabenazine and placebo groups, respectively (any AE, 59.5% vs 44.4-50.0% and 53.8%; treatment-related AE, 38.1% vs 18.1-25.0% and 30.8%). Serious AEs were reported for 2.8-8.3% of patients in the deutetrabenazine groups and 6.9% in the placebo group. Common AEs (≥ 4%) included headache, somnolence, nausea, anxiety, fatigue, dry mouth, and diarrhea. AE incidence was higher during the titration vs maintenance periods. For HD, AE incidence was numerically higher with deutetrabenazine (n = 84) vs placebo (n = 45; any AE, 64.3% vs 60.0%; treatment-related AE, 38.1% vs 26.7%); serious AEs were reported for similar proportions for the deutetrabenazine and placebo groups, 2.4% and 2.2%, respectively. Common AEs (≥ 4%) included irritability, fall, depression, dry mouth, and fatigue. CONCLUSIONS: Data from an integrated analysis of studies in TD and an integrated analysis of studies of chorea in HD showed that deutetrabenazine has a favorable safety profile and is well tolerated across indications. TRIAL REGISTRATION: ClinicalTrials.gov identifiers, NCT02291861, NCT02195700, NCT01795859, NCT02198794, NCT01897896.

Unintended movements are often the first sign of Huntington disease. This type of unintended movement is called chorea in Huntington disease. Tardive dyskinesia causes unintended body movements. Deutetrabenazine is a medicine used to treat both types of movements. This report summarizes deutetrabenazine safety across five clinical studies. Safety was assessed via adverse events (side effects). Adverse events were compared between deutetrabenazine and inactive treatment (placebo). Serious adverse events were also compared. Serious adverse events cause substantial impairment or disruption. In tardive dyskinesia and chorea in Huntington disease studies, most patients kept taking deutetrabenazine. Adverse events were not a common reason to stop treatment. For tardive dyskinesia, adverse event rates were similar between deutetrabenazine (≤ 60%) and placebo (54%). Serious adverse event rates were also similar for deutetrabenazine (≤ 8%) and placebo (7%). Adverse events tended to be reported earlier in treatment. Common adverse events were headache, sleepiness, nausea, anxiety, fatigue, dry mouth, and diarrhea. For chorea in Huntington disease, adverse event rates were similar for deutetrabenazine (64%) and placebo (60%). Serious adverse event rates were also similar for deutetrabenazine (2%) and placebo (2%). Irritability, fall, depression, dry mouth, and fatigue were common adverse events. Adverse events were similar between deutetrabenazine and placebo in both conditions. Deutetrabenazine was well tolerated for patients with either tardive dyskinesia or chorea in Huntington disease.

Fine needle aspiration of pancreatic lesions focusing on secondary tumors with emphasis of metastatic breast cancer: A clinicopathological study with follow-up.

BACKGROUND: The data on metastatic tumors to the pancreas diagnosed by fine needle aspiration (FNA) biopsy is limited. We report our experience of FNA of primary and secondary pancreatic tumors emphasizing metastatic breast cancer in the pancreas. METHOD: Total 274 cases of pancreatic FNA in 10 years were retrospectively reviewed. Literature review of metastatic breast cancers to the pancreas was performed. RESULTS: Out of the 274 cases, 7 (7/274, 2.6%) cases were non-diagnostic, 46 (46/274, 16.8%) cases were negative for malignancy, and 40 (40/274, 14.6%) cases were under the category of atypical cells. There were 133 (133/274, 48.5%) cases diagnosed as positive for malignancy, 20 (20/274, 7.3%) suspicious for malignancy, and 28 (28/274, 10.2%) cases in the category of neoplastic: other. The most common neoplasm diagnosed was ductal adenocarcinoma (114/274, 41.6%; 114/133, 85.7% in positive for malignancy category). Ten cases (10/274, 3.7%) were diagnosed as metastatic neoplasms to the pancreas, including four breast infiltrating ductal carcinomas (IDC), one endocervical adenocarcinoma, one anal/rectal squamous cell carcinoma, one renal cell carcinoma, one hepatocellular carcinoma, one seminoma and one lung adenocarcinoma. We summarized the biomarkers of the four metastatic breast cancers and conducted literature review on biomarkers of metastatic breast cancers to the pancreas. CONCLUSIONS: Upon analyzing FNAs of primary and secondary tumors in the pancreas, we have found breast carcinoma is the most common secondary pancreatic neoplasm in our patient population. Triple negative breast ductal carcinoma is the most common tumor among the metastasis of breast carcinomas to the pancreas. To the best of our knowledge, this study is the first report with a literature review focusing on biomarkers of metastatic breast cancer to the pancreas.

"Cattle Welfare Is Basically Human Welfare": Workers' Perceptions of 'Animal Welfare' on Two Dairies in China.

'Animal welfare' () is a foreign term in China, and stakeholder interpretations can affect receptiveness to the concept. Our aim was to explore workers' perceptions of animal welfare on two dairies in China. We used a mini-ethnographic case study design, with the first author (MC) living for 38 days on one farm and 23 days on a second farm. MC conducted semi-structured interviews (n = 13) and participant observations (n = 41) with farm management and staff. We used template analysis to generate key themes from the ethnographic data. Responses revealed a connection between human and animal welfare. Workers saw human welfare as a prerequisite to animal welfare, and cattle welfare as potentially mutually beneficial to humans. Some workers also saw an ethical obligation toward providing cattle with good welfare. Though some workers were unfamiliar with the term 'animal welfare,' in daily practice caring for cattle led farm workers to ponder, prioritize, and make decisions relevant to welfare including lameness, morbidity, and nutrition. Workers in management positions appeared to embrace evidence-based animal care improvements, especially those which were perceived to also benefit people. Based on our findings, we suggest animal welfare initiatives should (1) consider worker welfare, (2) clearly communicate the concept of 'animal welfare,' (3) identify mutual benefits, and (4) provide pragmatic, evidence-based strategies to improve welfare.

N-terminal serine/threonine motif has diverse and important effects on behavior of multiple AAV serotypes.

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.

Employee Management and Animal Care: A Comparative Ethnography of Two Large-Scale Dairy Farms in China.

Farm management can directly and indirectly affect animal care. We explored how farm management affected animal care on two large dairy farms in China (anonymized as Farm A and Farm B). We used a mini-ethnographic case study design whereby the first author lived for 38 days on Farm A and 23 days on Farm B. She conducted participant observation and ethnographic interviews with farm staff positions within five departments in Farm A and six departments in Farm B. In addition, she conducted 13 semi-structured interviews (seven on Farm A; six on Farm B). We used template analysis to generate key themes. On both farms, workers believed that animal care practices had improved over time, due to three key employee management factors: 1) organizational culture, 2) competency of worker and management, and 3) an effective incentive system. Our results suggest that animal care may be improved in this context by: 1) promoting a culture in which workers have 'grit' and are eager to learn, 2) ensuring basic worker wellbeing, and 3) using animal care outcomes as performance indicators linked to pay.

Display of Self-Peptide on Adeno-Associated Virus Capsid Decreases Phagocytic Uptake in Vitro.

Adeno-associated virus (AAV) vectors are currently investigated as gene transfer agents for the treatment of a variety of diseases. However, activation of the host immune response upon vector administration limits the use of AAV in the clinical setting. To decrease host detection of AAVs, we tested the CD47-based "don't-eat-me" signal in the context of the AAV capsid. We genetically incorporated the bioactive region of CD47, named "self-peptide" (SP), onto the surface of the AAV2 capsid. AAV mutants were structurally and functionally characterized for vector production, SP and linker incorporation into the capsid, transduction efficiency, and phagocytic susceptibility. We demonstrate that utilizing linkers improves the AAV2 capsid's tolerance to SP insertion. Notably, the SP significantly decreases the phagocytic susceptibility of AAV2 in vitro. Collectively, these results suggest that display of the SP motif on the AAV capsid surface can inhibit phagocytosis of the vector in vitrovia the "don't-eat-me" signaling.

Site-Specific Post-translational Surface Modification of Adeno-Associated Virus Vectors Using Leucine Zippers.

Adeno-associated virus (AAV) is widely favored as a gene therapy vector, tested in over 200 clinical trials internationally. To improve targeted delivery a variety of genetic capsid modifications, such as insertion of targeting proteins/peptides into the capsid shell, have been explored with some success but larger insertions often have unpredictable deleterious impacts on capsid formation and gene delivery. Here, we demonstrate a modular platform for the integration of exogenous peptides and proteins onto the AAV capsid post-translationally while preserving vector functionality. We decorated the AAV capsid with leucine-zipper coiled-coil binding motifs that exhibit specific noncovalent heterodimerization. AAV capsids successfully display hexahistidine tagged-peptides using this approach, as demonstrated through nickel column affinity. This protein display platform may facilitate the incorporation of biological moieties on the AAV surface, expanding possibilities for vector enhancement and engineering.

Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices.

The fields of physical, chemical, and synthetic virology work in partnership to reprogram viruses as controllable nanodevices. Physical virology provides the fundamental biophysical understanding of how virus capsids assemble, disassemble, display metastability, and assume various configurations. Chemical virology considers the virus capsid as a chemically addressable structure, providing chemical pathways to modify the capsid exterior, interior, and subunit interfaces. Synthetic virology takes an engineering approach, modifying the virus capsid through rational, combinatorial, and bioinformatics-driven design strategies. Advances in these three subfields of virology aim to develop virus-based materials and tools that can be applied to solve critical problems in biomedicine and biotechnology, including applications in gene therapy and drug delivery, diagnostics, and immunotherapy. Examples discussed include mammalian viruses, such as adeno-associated virus (AAV), plant viruses, such as cowpea mosaic virus (CPMV), and bacterial viruses, such as Qß bacteriophage. Importantly, research efforts in physical, chemical, and synthetic virology have further unraveled the design principles foundational to the form and function of viruses. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Longer Inactivating Sequence in Peptide Lock Improves Performance of Synthetic Protease-Activatable Adeno-Associated Virus.

Adeno-associated viruses (AAVs) are promising gene therapy vectors but may exhibit off-target delivery due to broad tissue tropism. We recently developed a synthetic protease-activatable AAV vector, named provector, that transduces cells preferentially in environments rich in matrix metalloproteinases (MMPs) which are elevated in a variety of diseases, including various cancers and heart diseases. The provector displays peptide locks made up of MMP recognition sites flanking an inactivating sequence (IS) composed of four aspartic acid residues (D4). When present, the IS prevents AAV from binding cell receptors and no transduction occurs (OFF state). High levels of MMPs cleave the recognition sequences and release the IS from the capsid surface, restoring cell receptor binding (ON state). The AAV9 provector prototype is not optimal as it displays baseline OFF transduction at 5-10% of that of the wild-type capsid, which can lead to off-target delivery. We hypothesized that changes to the IS may decrease OFF state transduction. We created a provector panel with IS of lengths 0 (D0) to 10 (D10) aspartic acid residues and characterized this panel in vitro. Notably, we find that the D10 provector has an OFF transduction of less than 1% of wild-type capsid and an ON/OFF transduction ratio of 27, the best outcome achieved for any provector thus far. In summary, our results enable us to define new design rules for the provector platform, specifically that (1) the IS is necessary for provector locking and (2) increasing the number of aspartic acid residues in this sequence improves locking.

Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA.

Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3.

Lasso peptides are a class of knot-like polypeptides in which the C-terminal tail of the peptide threads through a ring formed by an isopeptide bond between the N-terminal amine group and a side chain carboxylic acid. The small size (∼20 amino acids) and simple topology of lasso peptides make them a good model system for studying the unthreading of entangled polypeptides, both with experiments and atomistic simulation. Here, we present an in-depth study of the thermal unthreading behavior of two lasso peptides astexin-2 and astexin-3. Quantitative kinetics and energetics of the unthreading process were determined for variants of these peptides using a series of chromatography and mass spectrometry experiments and biased molecular dynamics (MD) simulations. In addition, we show that the Tyr15Phe variant of astexin-3 unthreads via an unprecedented "tail pulling" mechanism. MD simulations on a model ring-thread system coupled with machine learning approaches also led to the discovery of physicochemical descriptors most important for peptide unthreading.

Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.

OBJECTIVE: Characterization of the effects of HIV-1 infection and apoptosis on cellular and viral gene expression. METHODS: Flow cytometry was used to analyze infection and apoptosis concurrently in HIV-1IIIB-infected CEM-SS T cells. Suppression subtractive hybridization (SSH) was applied to cells from different time points of infection to construct subtracted complementary DNA (cDNA) libraries. Differential screening and Northern blots confirmed differential gene expression and these genes were sequenced and compared with database. RESULTS: T cells undergo apoptosis at early stages of HIV-1IIIB infection (days 5-7 post-infection). Surprisingly, cells begin to recover after day 9 and by day 18 almost all infected cells are viable, even though they maintain the same level of infection. By SSH, differential gene expression profiles between day 7 and day 18 after HIV-1IIIB infection were characterized. SSH yielded two subtracted cDNA libraries; differential screening of the subtracted cDNA libraries suggested that 200 out of 864 colonies were highly expressed at their respective time points. DNA sequence analysis identified specific apoptosis-related genes, HIV-1 viral genes, and other candidate genes of interest. Northern blot analysis confirmed that some of these genes were expressed predominantly at the 'apoptotic' or 'non-apoptotic' time points. CONCLUSIONS: Known and novel cellular gene products have been identified that are directly (or inversely) correlated with apoptosis and may regulate cell death in HIV-1 infection. These results provide a framework for functional studies on the differentially expressed genes and may suggest novel therapeutic approaches for treatment of HIV-1-infected individuals.