8-Cl-Ado and 8-NH2-Ado synergize with venetoclax to target the methionine-MAT2A-SAM axis in acute myeloid leukemia.

Targeting the metabolic dependencies of acute myeloid leukemia (AML) cells is a promising therapeutical strategy. In particular, the cysteine and methionine metabolism pathway (C/M) is significantly altered in AML cells compared to healthy blood cells. Moreover, methionine has been identified as one of the dominant amino acid dependencies of AML cells. Through RNA-seq, we found that the two nucleoside analogs 8-chloro-adenosine (8CA) and 8-amino-adenosine (8AA) significantly suppress the C/M pathway in AML cells, and methionine-adenosyltransferase-2A (MAT2A) is one of most significantly downregulated genes. Additionally, mass spectrometry analysis revealed that Venetoclax (VEN), a BCL-2 inhibitor recently approved by the FDA for AML treatment, significantly decreases the intracellular level of methionine in AML cells. Based on these findings, we hypothesized that combining 8CA or 8AA with VEN can efficiently target the Methionine-MAT2A-S-adenosyl-methionine (SAM) axis in AML. Our results demonstrate that VEN and 8CA/8AA synergistically decrease the SAM biosynthesis and effectively target AML cells both in vivo and in vitro. These findings suggest the promising potential of combining 8CA/8AA and VEN for AML treatment by inhibiting Methionine-MAT2A-SAM axis and provide a strong rationale for our recently activated clinical trial.

Freedom comes at a cost?: An exploratory study on affordances' impact on users' perception of a social robot.

Along with the development of speech and language technologies, the market for speech-enabled human-robot interactions (HRI) has grown in recent years. However, it is found that people feel their conversational interactions with such robots are far from satisfactory. One of the reasons is the habitability gap, where the usability of a speech-enabled agent drops when its flexibility increases. For social robots, such flexibility is reflected in the diverse choice of robots' appearances, sounds and behaviours, which shape a robot's 'affordance'. Whilst designers or users have enjoyed the freedom of constructing a social robot by integrating off-the-shelf technologies, such freedom comes at a potential cost: the users' perceptions and satisfaction. Designing appropriate affordances is essential for the quality of HRI. It is hypothesised that a social robot with aligned affordances could create an appropriate perception of the robot and increase users' satisfaction when speaking with it. Given that previous studies of affordance alignment mainly focus on one interface's characteristics and face-voice match, we aim to deepen our understanding of affordance alignment with a robot's behaviours and use cases. In particular, we investigate how a robot's affordances affect users' perceptions in different types of use cases. For this purpose, we conducted an exploratory experiment that included three different affordance settings (adult-like, child-like, and robot-like) and three use cases (informative, emotional, and hybrid). Participants were invited to talk to social robots in person. A mixed-methods approach was employed for quantitative and qualitative analysis of 156 interaction samples. The results show that static affordance (face and voice) has a statistically significant effect on the perceived warmth of the first impression; use cases affect people's perceptions more on perceived competence and warmth before and after interactions. In addition, it shows the importance of aligning static affordance with behavioural affordance. General design principles of behavioural affordances are proposed. We anticipate that our empirical evidence will provide a clearer guideline for speech-enabled social robots' affordance design. It will be a starting point for more sophisticated design guidelines. For example, personalised affordance design for individual or group users in different contexts.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

Using social robots for language learning: are we there yet?

Along with the development of speech and language technologies and growing market interest, social robots have attracted more academic and commercial attention in recent decades. Their multimodal embodiment offers a broad range of possibilities, which have gained importance in the education sector. It has also led to a new technology-based field of language education: robot-assisted language learning (RALL). RALL has developed rapidly in second language learning, especially driven by the need to compensate for the shortage of first-language tutors. There are many implementation cases and studies of social robots, from early government-led attempts in Japan and South Korea to increasing research interests in Europe and worldwide. Compared with RALL used for English as a foreign language (EFL), however, there are fewer studies on applying RALL for teaching Chinese as a foreign language (CFL). One potential reason is that RALL is not well-known in the CFL field. This scope review paper attempts to fill this gap by addressing the balance between classroom implementation and research frontiers of social robots. The review first introduces the technical tool used in RALL, namely the social robot, at a high level. It then presents a historical overview of the real-life implementation of social robots in language classrooms in East Asia and Europe. It then provides a summary of the evaluation of RALL from the perspectives of L2 learners, teachers and technology developers. The overall goal of this paper is to gain insights into RALL's potential and challenges and identify a rich set of open research questions for applying RALL to CFL. It is hoped that the review may inform interdisciplinary analysis and practice for scientific research and front-line teaching in future.

Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome.

The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on human DNMT1. Functional studies in human and mouse cells further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that preferentially occurs at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.

Long non-coding RNA lncMGC mediates the expression of TGF-ß-induced genes in renal cells via nucleosome remodelers.

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-ß (TGF-ß), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-ß, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation-qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-ß-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-ß-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-ß-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.

Autoclave treatment fails to completely inactivate DLB alpha-synuclein seeding activity.

Synucleinopathies are characterized by the deposition of alpha-synuclein (α-syn) aggregates in brain tissue. Pathological α-syn aggregates propagate in a prion-like manner and display prion-like biochemical properties. Using RT-QuIC, we measured α-syn seeding activity from brains of Dementia with Lewy body (DLB) patients post autoclave. Here, we show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining 50% seeding dose (SD50) is more than 107/mg tissue. DLB brain samples autoclaved at 132 °C still revealed an SD50 of approximately 106/mg tissue. Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.

ACTR5 controls CDKN2A and tumor progression in an INO80-independent manner.

Epigenetic dysregulation of cell cycle is a hallmark of tumorigenesis in multiple cancers, including hepatocellular carcinoma (HCC). Nonetheless, the epigenetic mechanisms underlying the aberrant cell cycle signaling and therapeutic response remain unclear. Here, we used an epigenetics-focused CRISPR interference screen and identified ACTR5 (actin-related protein 5), a component of the INO80 chromatin remodeling complex, to be essential for HCC tumor progression. Suppression of ACTR5 activated CDKN2A expression, ablated CDK/E2F-driven cell cycle signaling, and attenuated HCC tumor growth. Furthermore, high-density CRISPR gene tiling scans revealed a distinct HCC-specific usage of ACTR5 and its interacting partner IES6 compared to the other INO80 complex members, suggesting an INO80-independent mechanism of ACTR5/IES6 in supporting the HCC proliferation. Last, our study revealed the synergism between ACTR5/IES6-targeting and pharmacological inhibition of CDK in treating HCC. These results indicate that the dynamic interplay between epigenetic regulators, tumor suppressors, and cell cycle machinery could provide novel opportunities for combinational HCC therapy.

Cross-species parallels in babbling: animals and algorithms.

A key feature of vocal ontogeny in a variety of taxa with extensive vocal repertoires is a developmental pattern in which vocal exploration is followed by a period of category formation that results in a mature species-specific repertoire. Vocal development preceding the adult repertoire is often called 'babbling', a term used to describe aspects of vocal development in species of vocal-learning birds, some marine mammals, some New World monkeys, some bats and humans. The paper summarizes the results of research on babbling in examples from five taxa and proposes a unifying definition facilitating their comparison. There are notable similarities across these species in the developmental pattern of vocalizations, suggesting that vocal production learning might require babbling. However, the current state of the literature is insufficient to confirm this suggestion. We suggest directions for future research to elucidate this issue, emphasizing the importance of (i) expanding the descriptive data and seeking species with complex mature repertoires where babbling may not occur or may occur only to a minimal extent; (ii) (quasi-)experimental research to tease apart possible mechanisms of acquisition and/or self-organizing development; and (iii) computational modelling as a methodology to test hypotheses about the origins and functions of babbling. This article is part of the theme issue 'Vocal learning in animals and humans'.

ß-Barrel proteins tether the outer membrane in many Gram-negative bacteria.

Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)1. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun's lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability4; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present5. We used a glycoproteomic analysis of PG to show that ß-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the ß-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.

Acceptability and Effectiveness of NHS-Recommended e-Therapies for Depression, Anxiety, and Stress: Meta-Analysis.

BACKGROUND: There is a disconnect between the ability to swiftly develop e-therapies for the treatment of depression, anxiety, and stress, and the scrupulous evaluation of their clinical utility. This creates a risk that the e-therapies routinely provided within publicly funded psychological health care have evaded appropriate rigorous evaluation in their development. OBJECTIVE: This study aims to conduct a meta-analytic review of the gold standard evidence of the acceptability and clinical effectiveness of e-therapies recommended for use in the National Health Service (NHS) in the United Kingdom. METHODS: Systematic searches identified appropriate randomized controlled trials (RCTs). Depression, anxiety, and stress outcomes at the end of treatment and follow-up were synthesized using a random-effects meta-analysis. The grading of recommendations assessment, development, and evaluation approach was used to assess the quality of each meta-analytic comparison. Moderators of treatment effect were examined using subgroup and meta-regression analysis. Dropout rates for e-therapies (as a proxy for acceptability) were compared against controls. RESULTS: A total of 24 studies evaluating 7 of 48 NHS-recommended e-therapies were qualitatively and quantitatively synthesized. Depression, anxiety, and stress outcomes for e-therapies were superior to controls (depression: standardized mean difference [SMD] 0.38, 95% CI 0.24 to 0.52, N=7075; anxiety and stress: SMD 0.43, 95% CI 0.24 to 0.63, n=4863), and these small effects were maintained at follow-up. Average dropout rates for e-therapies (31%, SD 17.35) were significantly higher than those of controls (17%, SD 13.31). Limited moderators of the treatment effect were found. CONCLUSIONS: Many NHS-recommended e-therapies have not been through an RCT-style evaluation. The e-therapies that have been appropriately evaluated generate small but significant, durable, beneficial treatment effects. TRIAL REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO) registration CRD42019130184; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=130184.

Usability, Acceptability, and Effectiveness of Web-Based Conversational Agents to Facilitate Problem Solving in Older Adults: Controlled Study.

BACKGROUND: The usability and effectiveness of conversational agents (chatbots) that deliver psychological therapies is under-researched. OBJECTIVE: This study aimed to compare the system usability, acceptability, and effectiveness in older adults of 2 Web-based conversational agents that differ in theoretical orientation and approach. METHODS: In a randomized study, 112 older adults were allocated to 1 of the following 2 fully automated interventions: Manage Your Life Online (MYLO; ie, a chatbot that mimics a therapist using a method of levels approach) and ELIZA (a chatbot that mimics a therapist using a humanistic counseling approach). The primary outcome was problem distress and resolution, with secondary outcome measures of system usability and clinical outcome. RESULTS: MYLO participants spent significantly longer interacting with the conversational agent. Posthoc tests indicated that MYLO participants had significantly lower problem distress at follow-up. There were no differences between MYLO and ELIZA in terms of problem resolution. MYLO was rated as significantly more helpful and likely to be used again. System usability of both the conversational agents was associated with helpfulness of the agents and the willingness of the participants to reuse. Adherence was high. A total of 12% (7/59) of the MYLO group did not carry out their conversation with the chatbot. CONCLUSIONS: Controlled studies of chatbots need to be conducted in clinical populations across different age groups. The potential integration of chatbots into psychological care in routine services is discussed.

Altered distribution, aggregation, and protease resistance of cellular prion protein following intracranial inoculation.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

e-Therapies in England for stress, anxiety or depression: how are apps developed? A survey of NHS e-therapy developers.

OBJECTIVE: To document the quality of web and smartphone apps used and recommended for stress, anxiety or depression by examining the manner in which they were developed. DESIGN: The study was conducted using a survey sent to developers of National Health Service (NHS) e-therapies. DATA SOURCES: Data were collected via a survey sent out to NHS e-therapy developers during October 2015 and review of development company websites during October 2015. DATA COLLECTION/EXTRACTION METHODS: Data were compiled from responses to the survey and development company websites of the NHS e-therapies developers. RESULTS: A total of 36 (76.6%) out of the 48 app developers responded. One app was excluded due to its contact details and developer website being unidentifiable. Data from the missing 10 was determined from the app developer's website. The results were that 12 out of 13 web apps and 20 out of 34 smartphone apps had clinical involvement in their development. Nine out of 13 web apps and nine out of 34 smartphone apps indicated academic involvement in their development. Twelve out of 13 web apps and nine out of 34 smartphone apps indicated published research evidence relating to their app. Ten out of 13 web apps and 10 out of 34 smartphone apps indicated having other evidence relating to their app. Nine out of 13 web apps and 19 out of 34 smartphone apps indicated having a psychological approach or theory behind their app. CONCLUSIONS: As an increasing number of developers are looking to produce e-therapies for the NHS it is essential they apply clinical and academic best practices to ensure the creation of safe and effective apps.

Increases in theta CSD power and coherence during a calibrated stop-signal task: implications for goal-conflict processing and the Behavioural Inhibition System.

Psychologists have identified multiple different forms of conflict, such as information processing conflict and goal conflict. As such, there is a need to examine the similarities and differences in neurology between each form of conflict. To address this, we conducted a comprehensive electroencephalogram (EEG) analysis of Shadli, Glue, McIntosh, and McNaughton's calibrated stop-signal task (SST) goal-conflict task. Specifically, we examined changes in scalp-wide current source density (CSD) power and coherence across a wide range of frequency bands during the calibrated SST (n = 34). We assessed differences in EEG between the high and low goal-conflict conditions using hierarchical analyses of variance (ANOVAs). We also related goal-conflict EEG to trait anxiety, neuroticism, Behavioural Inhibition System (BIS)-anxiety and revised BIS (rBIS) using regression analyses. We found that changes in CSD power during goal conflict were limited to increased midfrontocentral theta. Conversely, coherence increased across 23 scalp-wide theta region pairs and one frontal delta region pair. Finally, scalp-wide theta significantly predicted trait neuroticism but not trait anxiety, BIS-anxiety or rBIS. We conclude that goal conflict involves increased midfrontocentral CSD theta power and scalp-wide theta-dominated coherence. Therefore, compared with information processing conflict, goal conflict displays a similar EEG power profile of midfrontocentral theta but a much wider coherence profile. Furthermore, the increases in theta during goal conflict are the characteristic of BIS-driven activity. Therefore, future research should confirm whether these goal-conflict effects are driven by the BIS by examining whether the effects are attenuated by anxiolytic drugs. Overall, we have identified a unique network of goal-conflict EEG during the calibrated SST.

Discriminating Between Imagined Speech and Non-Speech Tasks Using EEG.

People who are severely disabled (e.g Locked-in patients) need a communication tool translating their thoughts using their brain signals. This technology should be intuitive and easy to use. To this line, this study investigates the possibility of discriminating between imagined speech and two types of non-speech tasks related to either a visual stimulus or relaxation. In comparison to previous studies, this work examines a variety of different words with only single imagination in each trial. Moreover, EEG data are recorded from a small number of electrodes using a low-cost portable EEG device. Thus, our experiment is closer to what we want to achieve in the future as communication tool for locked-in patients. However, this design makes the EEG classification more challenging due to a higher level of noise and variations in EEG signals. Spectral and temporal features, with and without common spatial filtering, were used for classifying every imagined word (and for a group of words) against the non-speech tasks. The results show the potential for discriminating between each imagined word and non-speech tasks. Importantly, the results are different between subjects using different features showing the need for having subject specific features.

Processing of high-titer prions for mass spectrometry inactivates prion infectivity.

Prions represent a class of universally fatal and transmissible neurodegenerative disorders that affect humans and other mammals. The prion agent contains a pathologically aggregated form of the host prion protein that can transmit infectivity without any bacterial or viral component and is thus difficult to inactivate using disinfection protocols designed for infectious microorganisms. Methods for prion inactivation include treatment with acids, bases, detergents, bleach, prolonged autoclaving and incineration. During these procedures, the sample is often either destroyed or damaged such that further analysis for research purposes is compromised. In this study we show that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity, yielding a final product that fails to transmit prion disease in vivo. We further show that the resultant sample remains suitable for mass spectrometry-based protein identifications. Thus, the procedure described can be used to prepare prion-infected samples for mass spectrometry analysis with greatly reduced biosafety concerns.

Fibrinogen and the prediction of residual obstruction manifested after pulmonary embolism treatment.

Residual pulmonary vascular obstruction (RPVO) and chronic thromboembolic pulmonary hypertension (CTEPH) are both long-term complications of acute pulmonary embolism, but it is unknown whether RPVO can be predicted by variants of fibrinogen associated with CTEPH.We used the Akaike information criterion to select the best predictive models for RPVO in two prospectively followed cohorts of acute pulmonary embolism patients, using as candidate variables the extent of the initial obstruction, clinical characteristics and fibrinogen-related data. We measured the selected models' goodness of fit by analysis of deviance and compared models using the Chi-squared test.RPVO occurred in 29 (28.4%) out of 102 subjects in the first cohort and 46 (25.3%) out of 182 subjects in the second. The best-fit predictive model derived in the first cohort (p=0.0002) and validated in the second cohort (p=0.0005) implicated fibrinogen Bß-chain monosialylation in the development of RPVO. When the derivation procedure excluded clinical characteristics, fibrinogen Bß-chain monosialylation remained a predictor of RPVO in the best-fit predictive model (p=0.00003). Excluding fibrinogen characteristics worsened the predictive model (p=0.03).Fibrinogen Bß-chain monosialylation, a common structural attribute of fibrin, helped predict RPVO after acute pulmonary embolism. Fibrin structure may contribute to the risk of developing RPVO.

Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag.

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope "tags" to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways in vitro. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and in vivo imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics.