The McMaster Cough Severity Questionnaire (MCSQ): a cough severity instrument for patients with refractory chronic cough.

BACKGROUND: Cough severity represents an important endpoint to assess the impact of therapies for patients with refractory chronic cough (RCC). OBJECTIVE: To develop a new patient-reported outcome measure addressing cough severity in patients with RCC. METHODS: Phase 1 (item generation): A systematic survey, focus groups, and expert consultation generated 51 items. Phase 2 (item reduction): From a list of 51 items, 100 patients identified those they had experienced in the previous year and rated their importance on a 5-point scale. The MCSQ included items reported to occur most frequently and that had the highest importance scores. Patient feedback on the MCSQ led to elimination of redundant items. Another 100 patients completed the MCSQ, from which we performed an exploratory factor analysis and a Rasch analysis to further refine items on the MCSQ. RESULTS: Phase 2 led to selection of 15 items from the initial 51. Patient feedback on the 15 items led to elimination of 5 redundant items. An exploratory factor analysis of the 10-item MCSQ led to selection of two domains, elimination of one item that demonstrated cross-loading, and another that had high inter-item correlations. A Rasch analysis of the 8-item MCSQ confirmed that the response options functioned in a logically progressive manner and that no items exhibited differential item functioning. The final 8-item MCSQ has a one-week recall period and includes two domains (intensity and frequency). The 8-item MCSQ had high internal consistency (Cronbach's alpha, 0.89), proved able to distinguish different levels of cough severity (Pearson separation index, 0.89), and demonstrated high cross-sectional convergent validity (Pearson's correlation, 0.76 [95% CI 0.66 to 0.83]) with the 100-mm cough severity visual analogue scale. CONCLUSION: Initial evidence supports the validity of the MCSQ, an 8-item instrument measuring cough severity in patients with RCC. Future studies should evaluate its properties in measuring change over time.

Life at the conservative end of the leaf economics spectrum: intergeneric variation in the allocation of phosphorus to biochemical fractions in species of Banksia (Proteaceae) and Hakea (Proteaceae).

In severely phosphorus (P)-impoverished environments, plants have evolved to use P very efficiently. Yet, it is unclear how P allocation in leaves contributes to their photosynthetic P-use efficiency (PPUE) and position along the leaf economics spectrum (LES). We address this question in 10 species of Banksia and Hakea, two highly P-efficient Proteaceae genera. We characterised traits in leaves of Banksia and Hakea associated with the LES: leaf mass per area, light-saturated photosynthetic rates, P and nitrogen concentrations, and PPUE. We also determined leaf P partitioning to five biochemical fractions (lipid, nucleic acid, metabolite, inorganic and residual P) and their possible association with the LES. For both genera, PPUE was negatively correlated with fractional allocation of P to lipids, but positively correlated with that to metabolites. For Banksia only, PPUE was negatively correlated with residual P, highlighting a strategy contrasting to that of Hakea. Phosphorus-allocation patterns significantly explained PPUE but were not linked to the resource acquisition vs resource conservation gradient defined by the LES. We conclude that distinct P-allocation patterns enable species from different genera to achieve high PPUE and discuss the implications of different P investments. We surmise that different LES axes representing different ecological strategies coexist in extremely P-impoverished environments.

Discovery and first-time disclosure of CVN766, an exquisitely selective orexin 1 receptor antagonist.

Modulators of orexin receptors are being developed for neurological illnesses such as sleep disorders, addictive behaviours and other psychiatric diseases. We herein describe the discovery of CVN766, a potent orexin 1 receptor antagonist that has greater than 1000-fold selectivity for the orexin 1 receptor over the orexin 2 receptor and demonstrates low off target hits in a diversity screen. In agreement with its in vitro ADME data, CVN766 demonstrated moderate in vivo clearance in rodents and displayed good brain permeability and target occupancy. This drug candidate is currently being investigated in clinical trials for schizophrenia and related psychiatric conditions.

OTULIN limits cell death and inflammation by deubiquitinating LUBAC.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.

Differential Activities of the Botanical Extract PBI-05204 and Oleandrin on Innate Immune Functions under Viral Challenge Versus Inflammatory Culture Conditions.

The Nerium oleander extract PBI 05204 (PBI) and its cardiac glycoside constituent oleandrin have direct anti-viral properties. Their effect on the immune system, however, is largely unknown. We used an in vitro model of human peripheral blood mononuclear cells to document effects under three different culture conditions: normal, challenged with the viral mimetic polyinosinic:polycytidylic acid Poly I:C, and inflamed by lipopolysaccharide (LPS). Cells were evaluated for immune activation marks CD69, CD25, and CD107a, and culture supernatants were tested for cytokines. Both PBI and oleandrin directly activated Natural Killer (NK) cells and monocytes and triggered increased production of cytokines. Under viral mimetic challenge, PBI and oleandrin enhanced the Poly I:C-mediated immune activation of monocytes and NK cells and enhanced production of IFN-γ. Under inflammatory conditions, many cytokines were controlled at similar levels as in cultures treated with PBI and oleandrin without inflammation. PBI triggered higher levels of some cytokines than oleandrin. Both products increased T cell cytotoxic attack on malignant target cells, strongest by PBI. The results show that PBI and oleandrin directly activate innate immune cells, enhance anti-viral immune responses through NK cell activation and IFN-γ levels, and modulate immune responses under inflamed conditions. The potential clinical impact of these activities is discussed.

Accelerated embryonic stem cell screening with a highly efficient genotyping pipeline.

INTRODUCTION: Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. METHODS AND RESULTS: The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. Here, we optimized and functionally linked magnetic activated cell sorting (MACS) technology as well as multiplex droplet digital PCR (ddPCR) to our ES cell screening process to achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. We describe this improved workflow and illustrate the reduction in time between therapeutic target identification and experimental validation. CONCLUSION: In sum, we describe a novel and effective implementation of ddPCR, multiMACS, and adenovirus recombinase into a streamlined screening workflow that significantly reduces timelines for gene targeting in mouse ES cells.

Extracellular BMP1 is the major proteinase for COOH-terminal proteolysis of type I procollagen in lung fibroblasts.

Proteolytic processing of procollagens is a central step during collagen fibril formation. Bone morphogenic protein 1 (BMP1) is a metalloprotease that plays an important role in the cleavage of carboxy-terminal (COOH-terminal) propeptides from procollagens. Although the removal of propeptides is required to generate mature collagen fibrils, the contribution of BMP1 to this proteolytic process and its action site remain to be fully determined. In this study, using postnatal lung fibroblasts as a model system, we showed that genetic ablation of Bmp1 in primary murine lung fibroblasts abrogated COOH-terminal cleavage from type I procollagen as measured by COOH-terminal propeptide of type I procollagen (CICP) production. We also showed that inhibition of BMP1 by siRNA-mediated knockdown or small-molecule inhibitor reduced the vast majority of CICP production and collagen deposition in primary human lung fibroblasts. Furthermore, we discovered and characterized two antibody inhibitors for BMP1. In both postnatal lung fibroblast and organoid cultures, BMP1 blockade prevented CICP production. Together, these findings reveal a nonredundant role of extracellular BMP1 to process CICP in lung fibroblasts and suggest that development of antibody inhibitors is a viable pharmacological approach to target BMP1 proteinase activity in fibrotic diseases.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Susceptibility of canine chondrocytes and synoviocytes to antibiotic cytotoxicity in vitro.

OBJECTIVE: To evaluate relative cytotoxicity of antibiotics to normal canine joint tissues in vitro. STUDY DESIGN: Experimental in vitro study. SAMPLE POPULATION: Chondrocytes and synoviocytes (three dogs); cartilage explants (three dogs); six dogs total. METHODS: Chondrocytes and synoviocytes from normal femoropatellar joints of three dogs were plated on 24-well plates (50 000 cells/cm2 , triplicate, 48 hours) and exposed to antibiotics (ampicillin sulbactam, vancomycin, cefazolin, ceftazidime, amikacin, enrofloxacin; 0.39-25 mg/mL, 24 hours). Viability was assessed by using trypan blue dye exclusion. Antibiotic concentrations at which 50% cell death occurred (half-maximal inhibitory concentration) were determined to rank antibiotics for relative cytotoxicity. Occurrence of caspase-3 expression after antibiotic exposure was assessed as an indication of apoptosis induction. Cartilage explants from three different dogs were minced and exposed to antibiotics (amikacin, ceftazidime, cefazolin, enrofloxacin; 5 mg/mL, 72 hours). Live/dead staining was performed, and fluorescence was visualized by using confocal microscopy. Percentage of live vs dead cells was quantitated. RESULTS: Viability of chondrocytes and synoviocytes decreased with increasing antibiotic concentrations. Half-maximal inhibitory concentrations were determined for synoviocytes (vancomycin 13.77, ampicillin sulbactam 3.07, amikacin 2.26, ceftazidime 1.62, cefazolin 1.48, enrofloxacin 1.25 mg/mL) and chondrocytes (vancomycin 8.65, ampicillin sulbactam 8.63, ceftazidime 3.16, amikacin 2.74, cefazolin 1.67, enrofloxacin 0.78 mg/mL). Caspase-3 expression was upregulated, providing evidence that apoptotic pathways were active in cell death. CONCLUSION: Half-maximal inhibitory concentration data provided evidence of lower toxicity of vancomycin and ampicillin sulbactam to joint tissues in vitro. CLINICAL SIGNIFICANCE: These results provide evidence to justify future in vitro work with osteoarthritic joint tissues and in vivo clinical trials to evaluate safety and efficacy of intra-articular antibiotics to treat dogs with septic arthritis.

DeepSuccinylSite: a deep learning based approach for protein succinylation site prediction.

BACKGROUND: Protein succinylation has recently emerged as an important and common post-translation modification (PTM) that occurs on lysine residues. Succinylation is notable both in its size (e.g., at 100 Da, it is one of the larger chemical PTMs) and in its ability to modify the net charge of the modified lysine residue from + 1 to - 1 at physiological pH. The gross local changes that occur in proteins upon succinylation have been shown to correspond with changes in gene activity and to be perturbed by defects in the citric acid cycle. These observations, together with the fact that succinate is generated as a metabolic intermediate during cellular respiration, have led to suggestions that protein succinylation may play a role in the interaction between cellular metabolism and important cellular functions. For instance, succinylation likely represents an important aspect of genomic regulation and repair and may have important consequences in the etiology of a number of disease states. In this study, we developed DeepSuccinylSite, a novel prediction tool that uses deep learning methodology along with embedding to identify succinylation sites in proteins based on their primary structure. RESULTS: Using an independent test set of experimentally identified succinylation sites, our method achieved efficiency scores of 79%, 68.7% and 0.48 for sensitivity, specificity and MCC respectively, with an area under the receiver operator characteristic (ROC) curve of 0.8. In side-by-side comparisons with previously described succinylation predictors, DeepSuccinylSite represents a significant improvement in overall accuracy for prediction of succinylation sites. CONCLUSION: Together, these results suggest that our method represents a robust and complementary technique for advanced exploration of protein succinylation.

A Phase II, Single-Arm, Open-Label, Bayesian Adaptive Efficacy and Safety Study of PBI-05204 in Patients with Stage IV Metastatic Pancreatic Adenocarcinoma.

LESSONS LEARNED: This trial evaluating a novel plant extract, PBI-05204, did not meet its primary endpoint of overall survival but did show signals of efficacy in heavily pretreated mPDA. PBI-05204 was generally well tolerated, with the most common side effects related to treatment being vomiting (23.7%), nausea (18.4%), decreased appetite (18.4%), and diarrhea (15.8%). Additional trials are needed to explore the role of PBI-05204 in cancer treatment. BACKGROUND: Survival for metastatic pancreatic ductal adenocarcinoma (mPDA) is dismal, and novel agents are needed. PBI-05204 is a modified supercritical carbon dioxide extract of Nerium oleander leaves. Oleandrin, the extract's major cytotoxic component, is a cardiac glycoside that has demonstrated antitumor activity in various tumor cell lines with a mechanism involving inhibition of Akt phosphorylation and through downregulation of mTOR. METHODS: A phase II, single-arm, open-label study to determine the efficacy of PBI-05204 in patients with refractory mPDA therapy was conducted. The primary endpoint was overall survival (OS), with the hypothesis that 50% of patients would be alive at 4.5 months. Secondary objectives included safety, progression-free survival (PFS), and overall response rate. Patients received oral PBI-05204 daily until progressive disease (PD), unacceptable toxicity, or patient withdrawal. Radiographic response was assessed every two cycles. RESULTS: Forty-two patients were enrolled, and 38 were analyzed. Ten patients were alive at 4.5 months (26.3%) with a median PFS of 56 days. One objective response (2.6%) was observed for 162 days. Grade ≥ 3 treatment-emergent adverse events occurred in 63.2% of patients with the most common being fatigue, vomiting, nausea, decreased appetite, and diarrhea. CONCLUSION: PBI-05204 did not meet its primary endpoint for OS in this study. Recent preclinical data indicate a role for PBI-05204 against glioblastoma multiforme when combined with chemotherapy and radiotherapy. A randomized phase II trial is currently being designed.

Multimodality imaging of an azygous continuation of the caudal vena cava in a dog with pulmonary thromboembolic disease.

A 5-year-old spayed female English Bulldog was evaluated for acute anorexia, lethargy, respiratory distress, and syncope. Contrast-enhanced computed tomography revealed the vascular malformation of azygous continuation of the caudal vena cava with extensive thrombus formation and pulmonary arterial thromboembolic disease. The patient was hospitalized for supportive treatment and was prescribed long-term clopidogrel therapy. The patient survived to discharge and at last follow-up remained clinically stable. While this vascular malformation has been reported in canines, to the authors' knowledge, this is the first reported case of pulmonary thromboembolic disease in a canine concurrent with this condition.

An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.

The mechanics of spiral flow: Enhanced washout and transport.

Spiral/helical forms of blood flow have been observed in large arteries of the cardiovascular system, but their benefits remain underappreciated. Spiral flow has been postulated to improve near-wall washout, promoting anti-atherothrombotic conditions. This research aims to study the washout characteristics of spiral flow, specifically, its ability to increase velocity and wall shear stress (WSS) in atherothrombotic-prone regions. Using 1.2 cm diameter angled test-conduits (45°, 90°, 135°) with known recirculation/stasis regions at the bend corners, spiral flow washout potential was evaluated in terms of low velocity and low WSS. Two sub-studies were conducted: the first utilized a spiral flow-inducing device to enable qualitative analysis of washout-potential in both computational fluid dynamic (CFD) simulations and benchtop ultrasound visualization; the second used CFD to study the impact of several induced helical wavelengths on the conduit-dependent recirculation/stasis zones. Physical models of the angled conduits and spiral flow-inducer were 3D-printed to facilitate ultrasound visualization. Compared to straight flow, spiral flow generated by the flow-inducer significantly cleared the recirculation/stasis zones at the corners of the angled conduits. CFD simulations demonstrated that past a geometry-dependent threshold, increased helical content improved washout, denoted by decreased regions of low velocity and low WSS. Overall, spiral flow markedly improved washout in difficult to reach areas in the angled conduits. This has several important clinical implications: spiral flow shows great promise in reducing blood-transport-related complications and can be used to enhance the performance of future medical devices (eg grafts, mechanical circulatory support devices, hemodialysis access ports).

Complete overdenture fabrication for a 12-year-old child with dentinogenesis imperfecta type 2.

Dentinogenesis imperfecta type 2 (DI-2), also known as hereditary opalescent dentin, is a rare, genetically linked condition that affects both primary and permanent teeth. Severe attrition requiring full-mouth rehabilitation is a common finding associated with DI-2. Dental rehabilitation options include a variety of invasive and noninvasive restorative techniques dictated by the age of the patient. Growth and development must be considered and may result in a restorative challenge for the dental practitioner, particularly when the patient in question is a child. This case report describes the fabrication of an overdenture to reestablish function, esthetics, and self-esteem in a 12-year-old patient. A 2-stage restorative treatment was followed by a satisfactory 6-month recall examination, indicating that the prostheses provided a successful outcome until more definitive restorative treatment can be accomplished in adulthood.

Efficient conditional knockout targeting vector construction using co-selection BAC recombineering (CoSBR).

A simple and efficient strategy for Bacterial Artificial Chromosome (BAC) recombineering based on co-selection is described. We show that it is possible to efficiently modify two positions of a BAC simultaneously by co-transformation of a single-stranded DNA oligo and a double-stranded selection cassette. The use of co-selection BAC recombineering reduces the DNA manipulation needed to make a conditional knockout gene targeting vector to only two steps: a single round of BAC modification followed by a retrieval step.

Methadone maintenance-lessons from two systems in China.

In Hong Kong, methadone maintenance treatment (MMT) was launched in the 1970s, almost 30 years before the counterpart programme's inauguration in Mainland China. Both were established in response to perceived public crises-addiction-related crime and HIV outbreak, respectively-and both are now regular services under two systems in the same country. Effectiveness of MMT in achieving the stated goals was evident in each case and provides useful lessons on strategies for dealing with the varied concerns related to illicit drug use. Today, with changing patterns of drug addiction, increasing competition for resources, and changing attitudes towards addiction and its treatment, the two MMT systems are confronted with similar challenges to achieve sustainability.

Heat shock protein vaccination and directed IL-2 therapy amplify tumor immunity rapidly following bone marrow transplantation in mice.

Tumor relapse is the primary cause of mortality in patients with hematologic cancers following autologous hematopoietic stem cell transplantation (HSCT). Vaccination early after HSCT can exploit both the state of lymphopenia and minimal residual disease for generating antitumor immunity. Here, multiple vaccinations using lymphoma cells engineered to secrete heat shock protein fusion gp96-Ig within 2 weeks of T cell-replete syngeneic HSCT led to cross-presentation and increased survival of lymphoma-bearing mice. To enhance vaccine efficacy, interleukin (IL)-2 was directed to predominantly memory phenotype CD8(+) T lymphocytes and natural killer (NK) cells via administration bound to anti-IL-2 monoclonal antibody clone S4B6 (IL-2S4B6). Combination therapy with gp96-Ig vaccination and coordinated infusions of IL-2S4B6 resulted in marked prolongation of survival, which directly correlated with ~500% increase in effector CD8(+) T-cell numbers. Notably, this dual regimen elicited large increases in both donor CD8(+) T and NK cells, but not CD4(+) T lymphocytes; the former 2 populations are essential for both vaccine efficacy and protection against opportunistic infections after HSCT. Indeed, IL-2S4B6-treated HSCT recipients infected with Listeria monocytogenes exhibited decreased bacterial levels. These preclinical studies validate a new strategy particularly well suited to the post-HSCT environment, which may augment adaptive and innate immune function in patients with malignant disease receiving autologous HSCT.