Exploration of culturable bacterial associates of aphids and their interactions with entomopathogens.

Aphids shelter several bacteria that benefit them in various ways. The associates having an obligatory relationship are non-culturable, while a few of facultative associates are culturable in insect cell lines, axenic media or standard microbiology media. In the present investigation, isolation, and characterization of the culturable bacterial associates of various aphid species, viz., Rhopalosiphum maidis, Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum, and Lipaphis erysimi pseudobrassicae were carried out. A total of 42 isolates were isolated using different growth media, followed by their morphological, biochemical, and molecular characterization. The isolated culturable bacterial associates were found to belong to the genera Acinetobacter, Bacillus, Brevundimonas, Cytobacillus, Fictibacillus, Planococcus, Priestia, Pseudomonas, Staphylococcus, Sutcliffiella, and Tumebacillus which were grouped under seven families of four different orders of phyla Bacillota (Firmicutes) and Pseudomonata (Proteobacteria). Symbiont-entomopathogen interaction study was also conducted, in which the quantification of colony forming units of culturable bacterial associates of entomopathogenic fungal-treated aphids led us to the assumption that the bacterial load in aphid body can be altered by the application of entomopathogens. Whereas, the mycelial growth of entomopathogens Akanthomyces lecanii and Metarhizium anisopliae was found uninhibited by the bacterial associates obtained from Sitobion avenae and Rhopalosiphum padi. Analyzing persistent aphid microflora and their interactions with entomopathogens enhances our understanding of aphid resistance. It also fosters the development of innovative solutions for agricultural pest management, highlighting the intricate dynamics of symbiotic relationships in pest management strategies.

Lanthanum Cerate Microspheres for Efficient Fluoride Removal from Wastewater.

The performance of lanthanum cerate microspheres (LCM) at removing fluoride was analyzed in batch experiments after they were synthesized via the hydrothermal strategy. The ball-shaped microsphere morphology of LCM is confirmed by SEM and TEM. The synthesized LCM adsorbent showed excellent adsorption capacity in the pH range 3.0-7.0, with the optimal pH range being 3.5-4.5. The Langmuir adsorption model was more appropriate than the Freundlich model for describing the adsorption isotherm. The LCM adsorbent exhibited a significantly higher Langmuir adsorption capacity of 104.83 mg/g at pH 4.0, surpassing that of any other reported adsorbent. We investigated the adsorption of fluoride under a variety of conditions, including the presence of distinct anions. Furthermore, testing the adsorbent in actual groundwater demonstrated its high effectiveness in removing fluoride. Different useful analytical techniques were used for measurements and to learn and deduce the adsorption mechanism.

Specific Targeting and Labeling of Colonic Polyps in CPC-APC Mice with Mucin 5AC Fluorescent Antibodies: A Model for Detection of Early Colon Cancer.

Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 µg PBR was 1.70 (±0.56); the mean 50 µg PBR was 2.64 (±0.97); the mean 100 µg PBR was 3.32 (±1.33); and the mean 150 µg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 µg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

MiR-592 activates the mTOR kinase, ERK1/ERK2 kinase signaling and imparts neuronal differentiation signature characteristic of Group 4 medulloblastoma.

Medulloblastoma, a common malignant brain tumor in children, consists of four molecular subgroups WNT, SHH, Group 3 and Group 4. Group 3, Group 4 tumors have an overlap in their expression profiles and genetic alterations but differ significantly in their clinical characteristics, with Group 3 having the worst 5-year overall survival of <60%. MiR-592 is overexpressed predominantly in Group 4 tumors. MiR-592 expression reduced the anchorage-independent growth, invasion potential and tumorigenicity of Group 3 medulloblastoma cells. DEPTOR, an endogenous inhibitor of the mTOR kinase, and EML1 were identified as novel targets of miR-592. The miR-592 mediated decrease in the DEPTOR expression levels activated both mTORC1 and mTORC2 complex in medulloblastoma cells. However, the miR-592 expression also decreased the AKT kinase activity, likely to be due to the activation of the inhibitory feedback of the mTOR signaling. MiR-592 expression upregulated several neuronal differentiation-related genes, a characteristic of Group 4 medulloblastoma in Group 3 cell lines. The expression of miR-592 also upregulated the activity of ERK1/ERK2 kinases indicating activation of the MAPK signaling pathway. The inhibition of MAPK signaling by the ERK1/ERK2 inhibitor and mTOR signaling by rapamycin abrogated the miR-592-mediated upregulation of neuronal differentiation-related genes. Group 4 medulloblastomas showed higher activity of the mTOR and MAPK signaling compared to Group 3 tumors. Thus, miR-592 overexpression appears to be a driver event and a determining factor of Group 4 biology, which activates the mTOR and MAPK signaling pathways and thereby imparts its characteristic expression profile of neuronal differentiation-related genes.

The potentialities of omics resources for millet improvement.

Millets are nutrient-rich (nutri-rich) cereals with climate resilience attributes. However, its full productive potential is not realized due to the lack of a focused yield improvement approach, as evidenced by the available literature. Also, the lack of well-characterized genomic resources significantly limits millet improvement. But the recent availability of genomic data and advancement in omics tools has shown its enormous potential to enhance the efficiency and precision faced by conventional breeding in millet improvement. The development of high throughput genotyping platforms based on next-generation sequencing (NGS) has provided a low-cost method for genomic information, specifically for neglected nutri-rich cereals with the availability of a limited number of reference genome sequences. NGS has created new avenues for millet biotechnological interventions such as mutation-based study, GWAS, GS, and other omics technologies. The simultaneous discovery of high-throughput markers and multiplexed genotyping platform has aggressively aided marker-assisted breeding for millet improvement. Therefore, omics technology offers excellent opportunities to explore and combine useful variations for targeted traits that could impart high nutritional value to high-yielding cultivars under changing climatic conditions. In millet improvement, an in-depth account of NGS, integrating genomics data with different biotechnology tools, is reviewed in this context.

Bio-transfer and bioaccumulation patterns of heavy metals in mine site-inhabiting ants and grasshoppers, across mine site restoration chronosequence.

Soil is known to serve as a significant sink for heavy metals in coal mine sites, thus also influence the plant and other organisms of that area. Hence, the presence of heavy metals in coal mine soil is of concern to land managers. Insects occupy different trophic positions in the food chains, thus many insect species accumulate large amounts of heavy metals in their bodies and this is a matter of concern. In the present study, we investigated biotransfer and bioaccumulation of heavy metals from soil, grass species Cynodon dactylon, Vetiveria zizanioides, grasshopper species Gastrimargus africanus, Choroedocus robustus, ant species Cataglyphis longipedem and Camponotus compressus in six different ages (2, 4, 6, 8, 10 and 12 year old) of coal mine sites. Our study revealed that at some extent the heavy metal content and BAF patterns of heavy metals along different pathways (from soil to grass, soil to grasshoppers and from grass to grasshoppers) were not consistent and did not reflect the soil pollution status for all the metals, related to the mine spoil dump age. However, in contrast, ants successfully reflected a consistent pattern in the bioaccumulation of heavy metals via soil, thereby indicating the pollution status of the soil along with the restoration age of mine spoil dumps. Our study showed that ant species can successfully forecast the presence of heavy metals of coal mine spoils along with their restoration.

MmLwThV framework: A masked face periocular recognition system using thermo-visible fusion.

In wake of COVID-19, the world has adapted to a new order. People have started wearing mask on their faces to prevent getting infected. The present face recognition models are no longer proving to be efficient in the current circumstances. This is because, most of the informative part of the face is covered by mask. The periocular recognition therefore holds the key to future of face recognition. However, the periocular region proves to be insufficiently enough to generate highly discriminative features. Also, most of the pre-COVID-19 algorithms fail to work in cases, where the number of training images available is very less. We propose a lightweight periocular recognition framework that uses thermo-visible features and ensemble subspace network classifier to improve upon the existing periocular recognition systems named as Masked Mobile Lightweight Thermo-visible Face Recognition (MmLwThV). The framework successfully improves the accuracy over a single visible modality by mitigating the effect of noise present in the thermo-visible features. The experiments on WHU-IIP dataset and an in-house collected dataset named, CVBL masked dataset, successfully validate the efficacy of our proposed framework. The MmLwFR framework is lightweight and can be easily deployed on mobile phones with a visible and an infrared camera.

Artificial Intelligence-Based Assessment of Colorectal Polyp Histology by Elastic-Scattering Spectroscopy.

BACKGROUND: Colonoscopic screening and surveillance for colorectal cancer could be made safer and more efficient if endoscopists could predict histology without the need to biopsy and perform histopathology on every polyp. Elastic-scattering spectroscopy (ESS), using fiberoptic probes integrated into standard biopsy tools, can assess, both in vivo and in real time, the scattering and absorption properties of tissue related to its underlying pathology. AIMS: The objective of this study was to evaluate prospectively the potential of ESS to predict polyp pathology accurately. METHODS: We obtained ESS measurements from patients undergoing screening/surveillance colonoscopy using an ESS fiberoptic probe integrated into biopsy forceps. The integrated forceps were used for tissue acquisition, following current standards of care, and optical measurement. All measurements were correlated to the index pathology. A machine learning model was then applied to measurements from 367 polyps in 169 patients to prospectively evaluate its predictive performance. RESULTS: The model achieved sensitivity of 0.92, specificity of 0.87, negative predictive value (NPV) of 0.87, and high-confidence rate (HCR) of 0.84 for distinguishing 220 neoplastic polyps from 147 non-neoplastic polyps of all sizes. Among 138 neoplastic and 131 non-neoplastic polyps ≤ 5 mm, the model achieved sensitivity of 0.91, specificity of 0.88, NPV of 0.89, and HCR of 0.83. CONCLUSIONS: Results show that ESS is a viable endoscopic platform for real-time polyp histology, particularly for polyps ≤ 5 mm. ESS is a simple, low-cost, clinically friendly, optical biopsy modality that, when interfaced with minimally obtrusive endoscopic tools, offers an attractive platform for in situ polyp assessment.

Evaluation of phytoremediation capability of French marigold (Tagetes patula) and African marigold (Tagetes erecta) under heavy metals contaminated soils.

The pot experiment was conducted to explore the phytoremediation potential of two different marigold species grown in heavy metals contaminated red, black, alluvial, recent river clay, sewage irrigated, sewage sludge, and garden soil. Different soil types were treated uniformly with lead (20 mg Pb kg-1 soil), cadmium (5 mg Cd kg-1 soil), chromium (30 mg Cr kg-1 soil) and nickel (10 mg Ni kg-1 soil). Completely randomized design (CRD) was used with three replications. African marigold (Tagetes erecta) recorded ∼89.4% more dry matter yield over French marigold (Tagetes patula) when grown under metals treated soils. Both the marigold species were highly effective for removing Cd and Ni from contaminated soils (TF >1) however, Ni (TF ∼14.9) was more efficiently accumulated by T. patula and Cd (TF ∼12.1) by T. erecta. Higher biomass yield in T. erecta resulted higher accumulation of heavy metals (except Cr) compared to T. patula. Assessment of contamination factor (CF) and geo-accumulation index (Igeo) of heavy metals indicates that post-harvest soils had moderate to high degree of contamination by different metals, Cr being the highest. It may be concluded that T. erecta was more efficient in extracting heavy metals from different heavy metals contaminated soils.Novelty statement Contamination of land with heavy metals poses severe environmental threats. Physical and chemical remediation techniques are generally used for remediating metals contaminated sites. These methods are cost-intensive and therefore, commercially non-viable, besides being disruptive in nature and causing deterioration of soil. Alternatively, bio-remediation techniques are cost-effective and environment friendly. Among the various phytoremediation techniques, hyperaccumulator plants are most commonly used for the remediation of the contaminated sites. It has been found that different species of the same plant (marigold) differ in their ability to accumulate metals under various contaminated soils having different properties. Thus, this experiment provided an unique opportunity to investigate the effect of various soil properties on metal accumulation efficacy of marigold under metal-spiked soils. Marigold plants can grow rapidly by developing a robust root system which helps them to survive under contaminated soil environment. Thus, marigold being ornamental plant could be used to decontaminate polluted sites while providing ornamental value and may serve as a source of commercially valuable products extracting metals from biomass by way of incineration. However, meager information is available about the usage of various marigold species for phytoremediation of heavy metals under different metal-polluted soils. In the current experiment, we intend to evaluate the potential use of two different marigold species (Tagetes patula and Tagetes erecta) in remediating heavy metals under nine soils of different nature spiked with metals and assessing heavy metals pollution load indexes in these polluted soils.

Interaction of zinc oxide nanoparticles with soil: Insights into the chemical and biological properties.

Widespread use of zinc oxide nanoparticles (ZnO-NPs) threatens soil, plants, terrestrial and aquatic animals. Thus, it is essential to explore the fate and behavior of NPs in soil and also its mechanism of interaction with soil microbial biodiversity to maintain soil health and quality to accomplish essential ecosystem services. With this background, the model experiment was conducted in the greenhouse to study the impact of ZnO-NPs on soil taking maize as a test crop. The X-ray diffraction, Fourier transform infrared spectroscopy, Scanning electron microscopy and Particles size analysis of engineered NPs confirmed that the material was ZnO-NPs (particle size--65.82 nm). The application of ZnO-NPs resulted in a significant decrease in soil pH. Significantly high EC (0.13 dS m-1) was recorded where ZnO-NPs were applied at the rate of 2.5 mg Zn kg-1 soil over control (0.12 dS m-1). A significant increase in soil available phosphorus was observed on applying ZnO-NPs (15.29 mg kg-1 of soil) as compared to control (11.84 mg kg-1 of soil). Maximum soil available Zn (2.09 mg kg-1) was recorded in ZnO-NPs-amended soil (T11) which was significantly higher than control (0.33 mg kg-1) as well as treatments containing conventional zincatic fertilizers. The inhibition rates of dehydrogenase enzyme activity in the presence of 0.5 mg, 1.25 mg and 2.5 mg ZnO-NPs per kg soil were 31.3, 46.2 and 49.7%, respectively. Soil microbial biomass carbon was significantly reduced (103.33 µg g-1 soil) in soils treated with ZnO-NPs over control (111.33 µg g-1 soil). Soil bacterial count was also significantly lesser (12.33 × 105 CFU) in the case where 2.5 mg kg-1 ZnO-NPs were applied as compared to control (21.33 × 105 CFU). The corresponding decrease in fungal and actinomycetes colony count was 24.16, 37.35, 46.15% and 14.59, 17.97, 22.45% with the application of 0.5 mg, 1.25 mg and 2.5 mg ZnO-NPs per kg soil, respectively, as compared to control. Thus, the use of ZnO-NPs resulted in an increase in soil available Zn but inhibited soil microbial activity.

Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin.

Stasis of venous blood triggers deep vein thrombosis by activating coagulation, yet its effects on the fibrinolytic system are not fully understood. We examined the relationship between stasis, fibrinolysis, and the development of experimental venous thrombosis. Effects of stasis-induced deep vein thrombosis and fibrinolysis on thrombosis were examined by inferior vena cava ligation in congenic mice with and without α2-antiplasmin (α2AP), the primary inhibitor of plasmin. Venous thrombus weights were measured and thrombus composition was determined by Martius scarlet blue and immunofluorescence staining. Venous thrombi from α2AP+/+ mice contained plasminogen activators, plasminogen activator inhibitor-1, plasminogen, and α2AP, which changed with thrombus age. Normal, α2AP+/+ mice developed large, occlusive thrombi within 5 hours after ligation; thrombi were even larger in plasminogen-deficient mice (P < .001). No significant thrombus formation was seen in α2AP-/- mice (P < .0001) or in α2AP+/+ mice treated with an α2AP-inactivating antibody (P < .001). Venous stasis activated fibrinolysis, measured by D-dimer levels, in α2AP-/- mice vs α2AP+/+ mice (P < .05). Inhibition of fibrinolysis by the indirect plasmin inhibitor ε-aminocaproic acid or by α2AP restored thrombosis in α2AP-/- mice. In addition to its effects on acute thrombosis, thrombus formation was also markedly suppressed in α2AP-/- mice vs α2AP+/+ mice (P < .0001) 1, 7, and 14 days after ligation. We conclude that experimental venous stasis activates the fibrinolytic system to block the development of venous thrombosis. Suppression of fibrinolysis by α2AP appears essential for stasis-induced thrombus development, which suggests that targeting α2AP may prove useful for preventing venous thrombosis.

Real-time artificial intelligence-based histologic classification of colorectal polyps with augmented visualization.

BACKGROUND AND AIMS: Artificial intelligence (AI)-based computer-aided diagnostic (CADx) algorithms are a promising approach for real-time histology (RTH) of colonic polyps. Our aim is to present a novel in situ CADx approach that seeks to increase transparency and interpretability of results by generating an intuitive augmented visualization of the model's predicted histology over the polyp surface. METHODS: We developed a deep learning model using semantic segmentation to delineate polyp boundaries and a deep learning model to classify subregions within the segmented polyp. These subregions were classified independently and were subsequently aggregated to generate a histology map of the polyp's surface. We used 740 high-magnification narrow-band images from 607 polyps in 286 patients and over 65,000 subregions to train and validate the model. RESULTS: The model achieved a sensitivity of .96, specificity of .84, negative predictive value (NPV) of .91, and high-confidence rate (HCR) of .88, distinguishing 171 neoplastic polyps from 83 non-neoplastic polyps of all sizes. Among 93 neoplastic and 75 non-neoplastic polyps ≤5 mm, the model achieved a sensitivity of .95, specificity of .84, NPV of .91, and HCR of .86. CONCLUSIONS: The CADx model is capable of accurately distinguishing neoplastic from non-neoplastic polyps and provides a histology map of the spatial distribution of localized histologic predictions along the delineated polyp surface. This capability may improve interpretability and transparency of AI-based RTH and offer intuitive, accurate, and user-friendly guidance in real time for the clinical management and documentation of optical histology results.

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, kcat /Km , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.

Correction to: In Vivo Stability of Therapeutic Proteins.

A manuscript version without peer-review revisions was mistakenly processed and published.

In Vivo Stability of Therapeutic Proteins.

Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein. Stabilizing excipients used in protein formulations are expected to undergo more rapid distribution and dissociation in vivo, compared to a protein as a highly charged macromolecule. Thus, in vivo stability may significantly differ from shelf-life stability. In vivo degradation of the therapeutic protein may alter efficacy and/or safety characteristics such as immunogenicity. Studying the stability of a therapeutic protein in the intended body compartment can de-risk drug development in early stages of development by improving the selection of better clinical lead molecules. This review assesses the considerations when aiming to evaluate the in vivo fate of a therapeutic protein by comparing the physiology of relevant human body compartments and assessing their potential implications on the stability of a therapeutic protein. Moreover, we discuss the limitations of current experimental approaches mimicking physiologic conditions, depending on the desired route of administration, such as intravenous (IV), subcutaneous (SC), intravitreal (IVT), or intrathecal (IT) administration(s). New models more closely mimicking the relevant physiologic environment and updated analytical methods are required to understand the in vivo fate of therapeutic proteins.

A Semi-Physical Platform for Guidance and Formations of Fixed-Wing Unmanned Aerial Vehicles.

Unmanned Aerial Vehicles (UAVs) have multi-domain applications, fixed-wing UAVs being a widely used class. Despite the ongoing research on the topics of guidance and formation control of fixed-wing UAVs, little progress is known on implementation of semi-physical validation platforms (software-in-the-loop or hardware-in-the-loop) for such complex autonomous systems. A semi-physical simulation platform should capture not only the physical aspects of UAV dynamics, but also the cybernetics aspects such as the autopilot and the communication layers connecting the different components. Such a cyber-physical integration would allow validation of guidance and formation control algorithms in the presence of uncertainties, unmodelled dynamics, low-level control loops, communication protocols and unreliable communication: These aspects are often neglected in the design of guidance and formation control laws for fixed-wing UAVs. This paper describes the development of a semi-physical platform for multi-fixed wing UAVs where all the aforementioned points are carefully integrated. The environment adopts Raspberry Pi's programmed in C++, which can be interfaced to standard autopilots (PX4) as a companion computer. Simulations are done in a distributed setting with a server program designed for the purpose of routing data between nodes, handling the user inputs and configurations of the UAVs. Gazebo-ROS is used as a 3D visualization tool.

Computer-assisted assessment of colonic polyp histopathology using probe-based confocal laser endomicroscopy.

INTRODUCTION: Probe-based confocal laser endomicroscopy (pCLE) is a promising modality for classifying polyp histology in vivo, but decision making in real-time is hampered by high-magnification targeting and by the learning curve for image interpretation. The aim of this study is to test the feasibility of a system combining the use of a low-magnification, wider field-of-view pCLE probe and a computer-assisted diagnosis (CAD) algorithm that automatically classifies colonic polyps. METHODS: This feasibility study utilized images of polyps from 26 patients who underwent colonoscopy with pCLE. The pCLE images were reviewed offline by two expert and five junior endoscopists blinded to index histopathology. A subset of images was used to train classification software based on the consensus of two GI histopathologists. Images were processed to extract image features as inputs to a linear support vector machine classifier. We compared the CAD algorithm's prediction accuracy against the classification accuracy of the endoscopists. RESULTS: We utilized 96 neoplastic and 93 non-neoplastic confocal images from 27 neoplastic and 20 non-neoplastic polyps. The CAD algorithm had sensitivity of 95%, specificity of 94%, and accuracy of 94%. The expert endoscopists had sensitivities of 98% and 95%, specificities of 98% and 96%, and accuracies of 98% and 96%, while the junior endoscopists had, on average, a sensitivity of 60%, specificity of 85%, and accuracy of 73%. CONCLUSION: The CAD algorithm showed comparable performance to offline review by expert endoscopists and improved performance when compared to junior endoscopists and may be useful for assisting clinical decision making in real time.

Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation.

BACKGROUND: In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding. METHODS: Pulmonary embolism was induced by jugular vein infusion of 125I-fibrin or fluorescein isothiocyanate-fibrin labeled emboli in anesthetized mice. Thrombus site expression of key fibrinolytic molecules was determined by immunofluorescence staining. The effects of r-tPA and α2-antiplasmin inactivation on fibrinolysis and bleeding were examined in a humanized model of pulmonary embolism. RESULTS: The plasminogen activation and plasmin inhibition system assembled at the site of acute pulmonary emboli in vivo. Thrombus dissolution was markedly accelerated in mice with normal α2-antiplasmin levels treated with an α2-antiplasmin-inactivating antibody (P<0.0001). Dissolution of pulmonary emboli by α2-antiplasmin inactivation alone was comparable to 3 mg/kg r-tPA. Low-dose r-tPA alone did not dissolve emboli, but was synergistic with α2-antiplasmin inactivation, causing more embolus dissolution than clinical-dose r-tPA alone (P<0.001) or α2-antiplasmin inactivation alone (P<0.001). Despite greater thrombus dissolution, α2-antiplasmin inactivation alone, or in combination with low-dose r-tPA, did not lead to fibrinogen degradation, did not cause bleeding (versus controls), and caused less bleeding than clinical-dose r-tPA (P<0.001). CONCLUSIONS: Although the fibrinolytic system assembles at the site of pulmonary emboli, thrombus dissolution is halted by α2-antiplasmin. Inactivation of α2-antiplasmin was comparable to pharmacological r-tPA for dissolving thrombi. However, α2-antiplasmin inactivation showed a unique pattern of thrombus specificity, because unlike r-tPA, it did not degrade fibrinogen or enhance experimental bleeding. This suggests that modifying the activity of a key regulator of the fibrinolytic system, like α2-antiplasmin, may have unique therapeutic value in pulmonary embolism.

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen-Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.