PPIscreenML: Structure-based screening for protein-protein interactions using AlphaFold.

Protein-protein interactions underlie nearly all cellular processes. With the advent of protein structure prediction methods such as AlphaFold2 (AF2), models of specific protein pairs can be built extremely accurately in most cases. However, determining the relevance of a given protein pair remains an open question. It is presently unclear how to use best structure-based tools to infer whether a pair of candidate proteins indeed interact with one another: ideally, one might even use such information to screen amongst candidate pairings to build up protein interaction networks. Whereas methods for evaluating quality of modeled protein complexes have been co-opted for determining which pairings interact (e.g., pDockQ and iPTM), there have been no rigorously benchmarked methods for this task. Here we introduce PPIscreenML, a classification model trained to distinguish AF2 models of interacting protein pairs from AF2 models of compelling decoy pairings. We find that PPIscreenML out-performs methods such as pDockQ and iPTM for this task, and further that PPIscreenML exhibits impressive performance when identifying which ligand/receptor pairings engage one another across the structurally conserved tumor necrosis factor superfamily (TNFSF). Analysis of benchmark results using complexes not seen in PPIscreenML development strongly suggest that the model generalizes beyond training data, making it broadly applicable for identifying new protein complexes based on structural models built with AF2.

A novel intranasal peptide vaccine inhibits non-small cell lung cancer with KRAS mutation.

KRAS mutations occur commonly in the lung and can lead to the development of non-small cell lung cancer (NSCLC). While the mutated KRAS protein is a neoantigen, it usually does not generate an effective anti-tumor immune response on mucosal/epithelial surfaces. Despite this, mutated KRAS remains a potential target for immunotherapy since immune targeting of this protein in animal models has been effective at eliminating tumor cells. We attempted to develop a KRAS vaccine using mutated and wild-type KRAS peptides in combination with a nanoemulsion (NE) adjuvant. The efficacy of this approach was tested in an inducible mutant KRAS-mouse lung tumor model. Animals were immunized intranasally using NE with KRAS peptides. These animals had decreased CD4+FoxP3+ T cells in both lymph nodes and spleen. Immunized animals also showed higher IFN-γ and IL-17a levels to mutated KRAS that were produced by CD8+ T cells and enhancement in KRAS-specific Th1 and Th17 responses that persisted for 3 months after the last vaccination. Importantly, the immunized animals had significantly decreased tumor incidence compared to control animals. In conclusion, a mucosal approach to KRAS vaccination demonstrated the ability to induce local KRAS-specific immune responses in the lung and resulted in reduced tumor incidence.

Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Delicate Hybrid Laponite-Cyclic Poly(ethylene glycol) Nanoparticles as a Potential Drug Delivery System.

The objective of the study was to explore the feasibility of a new drug delivery system using laponite (LAP) and cyclic poly(ethylene glycol) (cPEG). Variously shaped and flexible hybrid nanocrystals were made by both the covalent and physical attachment of chemically homogeneous cyclized PEG to laponite nanodisc plates. The size of the resulting, nearly spherical particles ranged from 1 to 1.5 µm, while PEGylation with linear methoxy poly (ethylene glycol) (mPEG) resulted in fragile sheets of different shapes and sizes. When infused with 10% doxorubicin (DOX), a drug commonly used in the treatment of various cancers, the LAP-cPEG/DOX formulation was transparent and maintained liquid-like homogeneity without delamination, and the drug loading efficiency of the LAP-cPEG nano system was found to be higher than that of the laponite-poly(ethylene glycol) LAP-mPEG system. Furthermore, the LAP-cPEG/DOX formulation showed relative stability in phosphate-buffered saline (PBS) with only 15% of the drug released. However, in the presence of human plasma, about 90% of the drug was released continuously over a period of 24 h for the LAP-cPEG/DOX, while the LAP-mPEG/DOX formulation released 90% of DOX in a 6 h burst. The results of the cell viability assay indicated that the LAP-cPEG/DOX formulation could effectively inhibit the proliferation of A549 lung carcinoma epithelial cells. With the DOX concentration in the range of 1-2 µM in the LAP-cPEG/DOX formulation, enhanced drug effects in both A549 lung carcinoma epithelial cells and primary lung epithelial cells were observed compared to LAP-mPEG/DOX. The unique properties and effects of cPEG nanoparticles provide a potentially better drug delivery system and generate interest for further targeting studies and applications.

Wild-type SARS-CoV-2 neutralizing immunity decreases across variants and over time but correlates well with diagnostic testing.

Importance: The degree of immune protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants provided by infection versus vaccination with wild-type virus remains unresolved, which could influence future vaccine strategies. The gold-standard for assessing immune protection is viral neutralization; however, few studies involve a large-scale analysis of viral neutralization against the Omicron variant by sera from individuals infected with wild-type virus. Objectives: 1) To define the degree to which infection versus vaccination with wild-type SARS-CoV-2 induced neutralizing antibodies against Delta and Omicron variants.2) To determine whether clinically available data, such as infection/vaccination timing or antibody status, can predict variant neutralization. Methods: We examined a longitudinal cohort of 653 subjects with sera collected three times at 3-to-6-month intervals from April 2020 to June 2021. Individuals were categorized according to SARS-CoV-2 infection and vaccination status. Spike and nucleocapsid antibodies were detected via ADVIA Centaur® (Siemens) and Elecsys® (Roche) assays, respectively. The Healgen Scientific® lateral flow assay was used to detect IgG and IgM spike antibody responses. Pseudoviral neutralization assays were performed on all samples using human ACE2 receptor-expressing HEK-293T cells infected with SARS-CoV-2 spike protein pseudotyped lentiviral particles for wild-type (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants. Results: Vaccination after infection led to the highest neutralization titers at all timepoints for all variants. Neutralization was also more durable in the setting of prior infection versus vaccination alone. Spike antibody clinical testing effectively predicted neutralization for wild-type and Delta. However, nucleocapsid antibody presence was the best independent predictor of Omicron neutralization. Neutralization of Omicron was lower than neutralization of either wild-type or Delta virus across all groups and timepoints, with significant activity only present in patients that were first infected and later immunized. Conclusions: Participants having both infection and vaccination with wild-type virus had the highest neutralizing antibody levels against all variants and had persistence of activity. Neutralization of WT and Delta virus correlated with spike antibody levels against wild-type and Delta variants, but Omicron neutralization was better correlated with evidence of prior infection. These data help explain why 'breakthrough' Omicron infections occurred in previously vaccinated individuals and suggest better protection is observed in those with both vaccination and previous infection. This study also supports the concept of future SARS-CoV-2 Omicron-specific vaccine boosters.

Effective Treatment of Skin Wounds Co-Infected with Multidrug-Resistant Bacteria with a Novel Nanoemulsion.

Wound infections with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are particularly difficult to treat and present a great challenge to clinicians. Nanoemulsions (NE) are novel oil-in-water emulsions formulated from soybean oil, water, solvent, and surfactants such as benzalkonium chloride (BZK). An optimal ratio of those components produces nanometer-sized particles with the positive-charged surfactant at their oil-water interface. We sought to investigate antimicrobial NE as a novel treatment to address wounds co-infected by MRSA and VRE. Swine split-thickness skin wounds were first infected with MRSA and/or VRE, then treated with the nanoemulsion formulation (X-1735) or placebo controls. Bacterial viability after treatment were determined by nutrient agar plates for total, MRSA-specific, and VRE-specific loads. In addition, inflammation indexes were scored by histopathology. When VRE infected wounds were treated with X-1735, they contained 103 lower VRE CFU counts across a 2-week period compared with placebo. Once co-infected MRSA and VRE split-thickness wounds were successfully established, topical treatment of co-infected wounds with X-1735 resulted in a reduction of bacteria by 2 to 3 logs (compared with placebo) at 3- and 14-day postinfection time points. Importantly, X-1735 was effective in significantly alleviating multilevel inflammation in the treated wounds. X-1735 is a new antimicrobial that is safe to apply to open wounds and effectively kills MRSA and VRE. It appears to also reduce inflammation in these co-infected wounds. The data suggest that this approach offers promise as an antimicrobial for open wounds with MRSA and VRE co-infection. IMPORTANCE Infections, specifically polymicrobial, can cause serious consequences when it comes to wound treatment. Prolonged treatment with antibiotics can lead to an increased risk of bacterial resistance; co-infections can complicate treatment options even further. Our research proposes a novel nanoemulsion treatment for two of the most common antibiotic resistant bacteria: methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant enterococci (VRE). This optimized topical treatment formulation not only significantly reduces inflammation and infection in MRSA or VRE infected wounds, but also in MRSA and VRE co-infected wounds as well. The work aims to provide an alternative treatment approach for multidrug-resistant organisms and decrease dependence on systemic treatments.

Dendrimer-based posaconazole nanoplatform for antifungal therapy.

We examined formulating a new antifungal agent, posaconazole (POS) and its derivatives, with different molecular vehicles. Several combinations of drug and carrier molecules were synthesized, and their antifungal activities were evaluated against Aspergillus fumigatus. Posaconazole and four of its derivatives were conjugated to either generation 5 (G5) dendrimers or partially modified G5 dendrimers. The in vitro antifungal activities of these compounds suggest that conjugates with specific chemical linkages showed better fungistatic activity than direct conjugates to POS. In particular, a polyethylene glycol (PEG)-imidazole modified G5 dendrimer demonstrated improved antifungal efficacy relative to the parent G5 molecule. Further studies were then conducted with POS derived molecules coupled to PEG-imidazole modified G5 dendrimers to achieve a highly soluble and active conjugate of POS. This conjugated macromolecule averaged 23 POS molecules per G5 and had a high solubility with 50 mg/mL, which improved the molar solubility of POS from less than 0.03 mg/mL to as high as 16 mg/mL in water. The primary release profile of the drug in human plasma was extended to over 72 h, which is reflected in the in vitro inhibition of A. fumigatus growth of over 96 h. These POS-polymer conjugates appear to be novel and efficient antifungal agents.

Mild SARS-CoV-2 Illness Is Not Associated with Reinfections and Provides Persistent Spike, Nucleocapsid, and Virus-Neutralizing Antibodies.

Uncertainty exists whether mild COVID-19 confers immunity to reinfection. Questions also remain regarding the persistence of antibodies against SARS-CoV-2 after mild infection. We prospectively followed at-risk individuals with and without SARS-CoV-2 for reinfection and monitored the spike and nucleocapsid antibodies. This prospective cohort study was conducted over two visits, 3 to 6 months apart, between May 2020 and February 2021. Adults with and without COVID-19, verified by FDA EUA-approved SARS-CoV-2 RT-PCR assays, were screened for spike and nucleocapsid antibody responses using FDA EUA-approved immunoassays and for pseudoviral neutralization activity. The subjects were monitored for symptoms, exposure to COVID-19, COVID-19 testing, seroconversion, reinfection, and vaccination. A total of 653 subjects enrolled; 129 (20%) had a history of COVID-19 verified by RT-PCR at enrollment. Most had mild disease, with only three requiring hospitalization. No initially seropositive subjects experienced a subsequent COVID-19 infection during the follow-up versus 15 infections among initially seronegative subjects (infection rates of 0.00 versus 2.05 per 10,000 days at risk [P = 0.0485]). In all, 90% of SARS-CoV-2-positive subjects produced spike and nucleocapsid responses, and all but one of these had persistent antibody levels at follow-up. Pseudoviral neutralization activity was widespread among participants, did not decrease over time, and correlated with clinical antibody assays. Reinfection with SARS-CoV-2 was not observed among individuals with mild clinical COVID-19, while infections continued in a group without known prior infection. Spike and nucleocapsid COVID-19 antibodies were associated with almost all infections and persisted at stable levels for the study duration. IMPORTANCE This article demonstrates that people who have mild COVID-19 illnesses and produce antibodies are protected from reinfection for up to 6 months afterward. The antibodies that people produce in this situation are stable for up to 6 months as well. Clinical antibody assays correlate well with evidence of antibody-related viral neutralization activity.

Accurate point-of-care serology tests for COVID-19.

BACKGROUND: As COVID-19 vaccines become available, screening individuals for prior COVID-19 infection and vaccine response in point-of-care (POC) settings has renewed interest. We prospectively screened at-risk individuals for SARS-CoV-2 spike and nucleocapsid protein antibodies in a POC setting to determine if it was a feasible method to identify antibody from prior infection. METHODS: Three EUA-approved lateral flow antibody assays were performed on POC finger-stick blood and compared with serum and a CLIA nucleocapsid antibody immunoassay. Variables including antibody class, time since PCR, and the assay antigen used were evaluated. RESULTS: 512 subjects enrolled, of which 104 had a COVID-19 history and positive PCR. Only three PCR-positive subjects required hospitalization, with one requiring mechanical ventilation. The POC results correlated well with the immunoassay (93-97% sensitivity) and using serum did not improve the sensitivity or specificity. CONCLUSIONS: Finger-stick, POC COVID-19 antibody testing was highly effective in identifying antibody resulting from prior infections in mildly symptomatic subjects. Using high-complexity serum immunoassays did not improve the screening outcome. Almost all individuals with COVID-19 infection produced detectable antibodies to the virus. POC antibody testing is useful as a screen for prior COVID-19 infection, and should be useful in assessing vaccine response.

Outcome of PET-Negative Solid Pulmonary Nodules: A Retrospective Study.

OBJECTIVE: It was observed that malignancy had been found on follow-up in patients with PET-negative solid solitary pulmonary nodules (SPN). A retrospective analysis was performed to observe the natural history and malignant potential of these lesions, which, in routine practice, are presumed to be inactive. MATERIALS AND METHODS: Patients with an incidentally-discovered solid solitary pulmonary nodule who then had a negative follow-up PET/CT from 2005 to 2015 were identified using a text-based search methodology. These patients' charts were mined to determine the rate of development of subsequent malignancy from these index nodules. RESULTS: Of the patients with initially PET-negative solitary pulmonary nodule (n = 62, 43.5% women, mean age 65), 44 had clinical follow-up of the index lesion. 8 (7 pathology-proven) subsequent malignancies were identified with a mean time to diagnosis of 37.6 (±31.3) months. There were no statistically significant predictors of subsequent development of cancer (including age, gender, and smoking status). CONCLUSION: Upon follow up, 18.2% of the initially queried solid PET-negative nodules developed subsequent malignancy at an average time of 37.6 months, suggesting the continued need for follow-up of these initially PET-negative nodules beyond the 2 years currently suggested in popular guidelines. Importantly, these findings also remind radiologists that a negative PET/CT is not a surrogate for tissue diagnosis in the case of non-FDG avid SPN.

Functionalized nanoparticles with targeted antibody to enhance imaging of breast cancer in vivo.

BACKGROUND: Targeted contrast nanoparticles for breast tumor imaging facilitates early detection and improves treatment efficacy of breast cancer. This manuscript reports the development of an epidermal growth factor receptor-2 (HER-2) specific, bi-modal, dendrimer conjugate to enhance computed tomography (CT) and magnetic resonance imaging (MRI) of HER-2-positive breast cancer. This material employs generation 5 poly(amidoamine) dendrimers, encapsulated gold nanoparticles, chelated gadolinium, and anti-human HER-2 antibody to produce the nanoparticle contrast agent. RESULTS: Testing in two mouse tumor models confirms this contrast agent's ability to image HER-2 positive tumors. Intravenous injection of this nanoparticle in mice bearing HER-2 positive mammary tumors significantly enhances MRI signal intensity by ~ 20% and improves CT resolution and contrast by two-fold. Results by flow cytometry and confocal microscopy validate the specific targeting of the conjugate and its internalization in human HER-2 positive cells. CONCLUSION: These results demonstrate that this nanoparticle conjugate can efficiently target and image HER-2 positive tumors in vivo and provide a basis for the development of this diagnostic tool for early detection, metastatic assessment and therapeutic monitoring of HER-2 positive cancers.

A novel combination of intramuscular vaccine adjuvants, nanoemulsion and CpG produces an effective immune response against influenza A virus.

BACKGROUND: Vaccination is the most effective approach to prevent infection with highly pathogenic avian influenza (HPAI). Adjuvants are often used to induce effective immune responses and overcome the immunological weakness of recombinant HPAI antigens. Given the logistical challenges of immunization to HPAI during pandemic situations, vaccines administered via the intramuscular (I.M.) route would be of value. METHODS: A new formulation of nanoemulsion adjuvant (NE02) suitable for I.M. vaccination was developed. This NE02 was evaluated alone and in combination with CpG to develop H5 immune responses in mouse and ferret models. Measures of recombinant H5 (rH5) specific immunity evaluated included serum IgG and IgG subclasses, bronchoalveolar lavage fluid IgA, and cytokines. The activation of NF-kB was also analyzed. The efficacy of the vaccine was assessed by performing hemagglutination inhibition (HAI), virus neutralization (VN) assays, and viral challenges in ferrets. RESULTS: I.M. vaccination with rH5-NE02 significantly increased rH5-specific IgG and protected ferrets in the viral challenge model providing complete protection and sterile immunity in all animals tested. Combining NE02 and CpG produced accelerated antibody responses and this was accompanied by an elevation of IFN-γ and IL-17 responses and the downregulation of IL-5. The combination also caused a synergistic effect on NF-kB activation. In immunized ferrets after viral challenge, the rH5-NE02 + CpG vaccine via I.M. achieved at least 75% and 88% seroconversion of HAI and VN antibody responses, respectively, and improved body temperature stabilization and weight loss over NE02 alone. CONCLUSIONS: The I.M. injection of NE02 adjuvanted rH5 elicits strong and broad immune responses against H5 antigens and effectively protects animals from lethal H5 challenge. Combining this adjuvant with CpG enhanced immune responses and provided improvements in outcomes to viral challenge in ferrets. The results suggest that combinations of adjuvants may be useful to enhance H5 immune responses and improve protection against influenza infection.

Targeted protein degradation mechanisms.

Targeted protein degradation mediated by small molecule degraders represents an exciting new therapeutic opportunity to eliminate disease-causing proteins. These molecules recruit E3 ubiquitin ligases to the protein of interest and mediate its ubiquitination and subsequent proteolysis by the proteasome. Significant advancements have been made in the discovery and development of clinically relevant degraders. In this review we will focus on the recent progress in understanding ternary complex formation and structures, ubiquitination, and other critical factors that govern the efficiency of degraders both in vitro and in vivo. With deeper knowledges of these areas, the field is building guiding principles to reduce the level of empiricism and to identify therapeutically relevant degraders more rationally and efficiently.

Congenital absence of the internal carotid artery with intercavernous anastomosis.

Congenitally absent internal carotid artery with intercavernous anastomosis is an exceedingly rare vascular anomaly. We report such a case in a 65-year-old man with chronic symptoms resembling sporadic transient ischemic attacks. While these patients are usually asymptomatic due to compensatory collateralization, they are at increased risk of aneurysm formation, and thus proper identification is important. This report serves as both a case of a very rare anomaly and as a lesson on how to avoid this misdiagnosis by carotid duplex sonography. Additionally, we review the limited number of published cases of congenitally absent internal carotid artery with intercavernous anastomosis.

Bilirubin Correlation May Preclude MRCP in Acute Cholecystitis Patients With Normal Common Bile Duct Diameter.

OBJECTIVE. In patients with acute cholecystitis (AC), accurate identification of a common bile duct (CBD) stone before cholecystectomy is of concern for surgeons, gastroenterologists, and radiologists. This study evaluates the utility of preoperative MRCP taking into consideration both sonographic findings and biochemical predictors for choledocholithiasis. MATERIALS AND METHODS. Fifty-seven patients (58% women; mean age, 54 years old) with signs of AC on right upper quadrant (RUQ) ultrasound (US) who underwent subsequent MRCP from 2007 to 2017 were identified using a text-based search and retrospectively analyzed, using ERCP as the reference standard. RESULTS. For patients with AC who had a normal CBD diameter on initial RUQ US, we found a significant difference in the total and direct bilirubin levels of patients who had positive (1.94 vs 4.02 mg/dL, respectively; p = 0.013) and negative (0.71 vs 2.13 mg/dL, respectively; p = 0.02) findings for CBD stone on MRCP. ROC curve analysis showed an increased total bilirubin threshold of > 2.3 mg/dL (standard threshold, 1.2 mg/dL), which yielded a negative predictive value (NPV) of 95%. An increased direct bilirubin threshold of > 0.9 mg/dL (standard threshold, 0.2 mg/dL) yielded an NPV of 100%. CONCLUSION. In patients with AC who have a normal CBD diameter on RUQ US, normal or even mildly elevated bilirubin levels below a calculated threshold may obviate preoperative MRCP. Radiologists should be active participants in clinical decision-making; discussion between referring physicians and radiologists regarding biochemical markers and sonographic findings will lead to more appropriate use of preoperative imaging.

Recombinant H5 hemagglutinin adjuvanted with nanoemulsion protects ferrets against pathogenic avian influenza virus challenge.

BACKGROUND: Highly pathogenic H5N1 influenza viruses remain a pandemic risk to the world population. Although vaccines are the best solution to prevent this threat, a more effective vaccine for H5 strains of influenza has yet to be developed. All existing vaccines target only serum antibody against influenza as the primary outcome, while mucosal immunity has not been addressed. To address these shortcomings we have used an effective mucosal adjuvant system to produce a prototype vaccine that provides antibody, cellular and mucosal immunity to multiple serotypes of H5. METHODS: Plant-derived recombinant H5 (rH5) antigen was mixed with a novel nanoemulsion NE01 adjuvant. The rH5-NE01 vaccine was administered intranasally to CD-1 mice and ferrets. Immunogenicity of this immunization was evaluated through rH5-specific antibody and cellular immune responses. Hemagglutination inhibition (HI) and virus neutralization (VN) assays were performed. Protection against H5N1 virus challenge was evaluated in ferrets. RESULTS: Intranasal immunization with rH5-NE01vaccine induced high titers (>106) of rH5-specific IgG in mice. In mice and ferrets this vaccine also achieved titers of ≥40 for both HI and VN. Additionally, the levels of rH5-specific IgA were significantly increased in bronchial secretions in these animals. The rH5-NE01 vaccine enhanced rH5-specific cellular immune responses including IFN-γ and IL-17. Ten-day survival post challenge was 100% in ferrets that received rH5-NE01compared to 12.5% in the PBS group. Furthermore, this vaccine prevented weight loss and increases in body temperature after H5N1 challenge as compared to the controls. Moreover, H5N1 virus in nasal wash of rH5-NE01-vaccinated ferrets was significantly decreased compared to controls. CONCLUSION: Intranasal immunization with rH5 antigen formulated with NE01 adjuvant elicited strong, broad and balanced immune responses that effectively protect against H5N1 influenza virus infection in the ferret model. The ease of formulation of rH5-NE01 makes this novel combination a promising mucosal vaccine candidate for pandemic influenza.

Treatment of superior vena cava syndrome using AngioJet™ thrombectomy system.

BACKGROUND: Superior vena cava syndrome is a relatively rare presentation in which diminished venous return to the heart produces congestion of the neck, face and upper extremities. Typically, a mediastinal mass produces external compression on the superior vena cava and reduces venous return. However, superior vena cava syndrome can present acutely in the setting of vena cava thrombosis. Multiple scoring systems are available to assist clinicians with appropriate timing of interventions for SVC syndrome. When specific criteria are met, endovascular intervention can be beneficial to patients to prevent rapid deterioration. CASE PRESENTATION: A 75-year-old female with no significant past medical history presented to the emergency department with increased facial swelling, nausea and vomiting which began the night prior to presentation. The patient underwent a CT chest which revealed a 3.2 × 3.0 × 3.8 cm spiculated mass compressing the right main bronchus and right pulmonary artery. The patient was intubated and interventional radiology was consulted. The patient underwent venography which showed extensive thrombosis of the innominate veins. Rheolytic thrombectomy with AngioJet™ was performed to alleviate clot burden and minimize risk of secondary pulmonary embolism. Kissing stents were placed in the bilateral innominate veins to maintain patency after thrombectomy. After the procedure, the patient was successfully extubated and had near complete resolution of facial swelling approximately 12 h post procedure. A follow up venogram performed on post procedure day 4 showed patent bilateral subclavian, innominate, and internal jugular veins as well as a patent superior vena cava. CONCLUSIONS: Acute occlusion of superior vena cava can present with life threatening symptoms such as loss of airway. AngioJet™ thrombectomy is another tool available to interventional radiologists when a patient's clinical condition necessitates treatment.

Replaced gastroduodenal artery with continuation as accessory left hepatic artery: a rare anatomical variant.

BACKGROUND: Presented here is a rare case of a gastroduodenal artery (GDA) replaced to the superior mesenteric artery with continuation into an accessory left hepatic artery. To the authors' knowledge, this rare anomaly is not described previously. Replaced GDA is reported, however without continuation into an accessory LHA. CASE PRESENTATION: A 74-year-old male with recurrent peptic ulcer disease presented with acute onset melena. This anomaly was discovered during abdominal angiography performed for treatment of a hemorrhagic duodenal ulcer. Initial celiac and common hepatic artery (CHA) subselection and angiography demonstrated no branch vessels. Angiography via the superior mesenteric artery demonstrated free extravasation into the duodenum and distal perfusion of the left hepatic lobe. CONCLUSIONS: This is a rare variant of hepatic arterial anatomy. Importantly, this variant challenges a previously described anatomical model that attempts to explain observed CHA variation.

The afatinib resistance of in vivo generated H1975 lung cancer cell clones is mediated by SRC/ERBB3/c-KIT/c-MET compensatory survival signaling.

We generated afatinib resistant clones of H1975 lung cancer cells by transient exposure of established tumors to the drug and collected the re-grown tumors. Afatinib resistant H1975 clones did not exhibit any additional mutations in proto-oncogenes when compared to control clones. Afatinib resistant H1975 tumor clones expressed less PTEN than control clones and in afatinib resistant clones this correlated with increased basal SRC Y416, ERBB3 Y1289, AKT T308 and mTOR S2448 phosphorylation, decreased expression of ERBB1, ERBB2 and ERBB3 and increased total expression of c-MET, c-KIT and PDGFRß. Afatinib resistant clones were selectively killed by knock down of [ERBB3 + c-MET + c-KIT] but not by the individual or doublet knock down combinations. The combination of the ERBB1/2/4 inhibitor afatinib with the SRC family inhibitor dasatinib killed afatinib resistant H1975 cells in a greater than additive fashion; other drugs used in combination with dasatinib such as sunitinib, crizotinib and amufatinib were less effective. [Afatinib + dasatinib] treatment profoundly inactivated ERBB3, AKT and mTOR in the H1975 afatinib resistant clones and increased ATG13 S318 phosphorylation. Knock down of ATG13, Beclin1 or eIF2α strong suppressed killing by [ERBB3 + c-MET + c-KIT] knock down, but were only modestly protective against [afatinib + dasatinib] lethality. Thus afatinib resistant H1975 NSCLC cells rely on ERBB1- and SRC-dependent hyper-activation of residual ERBB3 and elevated signaling, due to elevated protein expression, from wild type c-MET and c-KIT to remain alive. Inhibition of ERBB3 signaling via both blockade of SRC and ERBB1 results in tumor cell death.

Mechanistic study of Uba5 enzyme and the Ufm1 conjugation pathway.

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.