Fusion Assay Research Articles

A novel GLCC1::BRAF fusion with independent MYCN and MYC amplifications in primary tumor and metastasis in pediatric pancreatic acinar carcinoma

Abstract Introduction/Objective Pediatric pancreatic acinar cell carcinoma (PACC) is a rare malignancy, comprising 5-15% of pediatric pancreatic tumors. BRAF rearrangement is found in 20-30% of PACC cases, most commonly an inversion in chromosome 7. We report a case of PACC with GLCC1::BRAF fusion. Methods/Case Report A 10-year-old male presented with six months of weight loss, back pain, and loose stools. Imaging demonstrated concentric soft tissue thickening around the celiac axis and superior mesenteric artery, clinically interpreted as vasculitis. Follow-up imaging showed extension of the soft tissue thickening, prompting biopsy of a periaortic lymph node showing metastatic PACC. Pancreaticduodenectomy revealed a 3.8 cm pancreatic neck mass with perineural invasion and metastatic deposits in multiple lymph nodes and retroperitoneal soft tissue. Fluorescence in situ hybridization of both specimens demonstrated BRAF gene rearrangement, with the partner identified as GLCC1 by next generation sequencing and fusion assays. Microarray on the lymph node demonstrated amplification of MYC on chromosome 8, but not in the resection specimen, which showed gain of chromosome 2 and amplification of MYCN. Results (if a Case Study enter NA) NA Conclusion The GLCC1::BRAF fusion has previously been reported once in a case of pheochromocytoma with peritoneal seeding. The MYC gene family members, c-MYC and MYCN, are involved many cancer types; amplifications are often associated with unfavorable prognosis in tumors such as neuroblastoma. However, the amplification of both genes in the same tumor at different loci has not been reported. The combination of the primary GLCC1::BRAF fusion with secondary amplification of MYC and MYCN is likely to drive the aggressive behavior and metastasis in this case of PACC. Treatment for PACC includes chemoradiation and BRAF inhibition. However, BRAF rearrangements without the V600E mutation show variable response to BRAF inhibitors. Potential alternatives include inhibitors to other targets within the BRAF signaling pathway. Further investigation is needed to determine the clinicopathologic impact of these novel genetic aberrancies.

RET Fusion Detection Using IdyllaTM Gene Fusion Assay

Abstract Introduction/Objective Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancers and RET fusions are detected in 1-2% of NSCLCs, 70% medullary thyroid cancers, 20% papillary thyroid cancers, and <1% of solid tumors. Next-generation sequencing (NGS) is frequently used, taking 2-3 weeks, and requires many tissue samples (10-20 formalin-fixed paraffin-embedded, FFPE, tissue sections). The Biocartis Idylla Gene Fusion Assay (IGFA), a rapid real-time walk-away RNA-RTPCR-based system, was used to detect RET, ALK, ROS1, NTRK 1/2/3 rearrangements, and MET exon 14 skipping mutations in a single assay. We hypothesized that the Idylla Gene Fusion Assay would improve patient care by detecting fusion gene(s) with 2 slides in 3 hours. Methods/Case Report This retrospective study compares NGS and IGFA using FFPE tissue from The Inova Health System (10 patients total). Two unstained 4-micron-thick sections were obtained per tissue block using aseptic techniques. RET fusion results from NGS and IGFA were compared, as well as turnaround time (TAT) and tissue requirements. Results (if a Case Study enter NA) Patient 3 had negative RET fusion by IGFA, but positive results using NGS. Patients 4, 5, and 6 had a positive correlation between IGFA and NGS. Patients 9, 10 and 11 had negative RET results by both IGFA and NGS, however Patient 10 IGFA results detected a positive NTRK3 mutation. Patient 7 was positive for RET mutation by IGFA, but passed away while awaiting NGS results. IGFA generated results in three hours while NGS had an average TAT of 14.82 days. Patients 1, 7, and 8 had insufficient remaining tissue for IGFA. IGFA Cq values were used for quality control. Conclusion RET fusions can be detected using the IGFA for timely treatment with limited tissue. This assay is an easy-to-use, walk-away, stand-alone diagnostic tool that can detect gene fusions on the same day, on demand while waiting for NGS results.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein

African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. Rapid and early diagnosis is crucial for the prevention of ASF. ASFV mature virions comprise the inner envelope protein, p22, making it an excellent candidate for the serological diagnosis and surveillance of ASF. In this study, the prokaryotic-expressed p22 recombinant protein was prepared and purified for immunization in mice. Four monoclonal antibodies (mAbs) were identified using hybridoma cell fusion, clone purification, and immunological assays. The epitopes of mAbs 14G1 and 22D8 were further defined by alanine-scanning mutagenesis. Our results showed that amino acids C39, K40, V41, D42, C45, G48, E49, and C51 directly bound to 14G1, while the key amino acid epitope for 22D8 included K161, Y162, G163, D165, H166, I167, and I168. Homologous and structural analysis revealed that these sites were highly conserved across Asian and European ASFV strains, and the amino acids identified were located on the surface of p22. Thus, our study contributes to a better understanding of the antigenicity of the ASFV p22 protein, and the results could facilitate the prevention and control of ASF.

Optical Genome Mapping Allows Detection and Characterization of Cytogenetically Cryptic Oncogenic Fusions in Pediatric Acute Myeloid Leukemia

Background: Abnormal gene fusions are predominant oncogenic drivers in pediatric acute myeloid leukemia (AML) and are important diagnostic and prognostic markers. However, several oncogenic fusions specific for or enriched in pediatric AML (like CBFA2T3::GLIS2, NUP98::NSD1, ETV6::MNX1 and others) are not detectable by conventional cytogenetic analysis (cytogenetically cryptic) and may not be included in fluorescence in situ hybridization (FISH) or molecular fusion panels designed for adult patients. Optical genome mapping (OGM) is a powerful new method for genome-wide, high-resolution detection of copy number abnormalities and balanced chromosome rearrangements. This study determined the frequency of cryptic oncogenic fusions in an institutional cohort of pediatric AML cases and evaluated OGM as a method for their detection and characterization. Methods: Retrospective review of genetic testing results was performed for 93 consecutive de novo or relapsed pediatric AML cases tested at the Children's Hospital Los Angeles (CHLA) Center for Personalized Medicine between March 2016 and July 2023. Genetic testing was performed as part of clinical care following standard procedures and consisted of karyotype analysis, FISH testing, chromosomal microarray (CMA) analysis, CHLA custom sequencing panel (OncoKids®) (June 2017 onwards) and RNA-Seq fusion assay (March 2022 onwards). Cases with normal or inconclusive karyotype in which an oncogenic fusion was detected by another clinical assay were selected for OGM testing. High molecular weight DNA from peripheral blood or bone marrow samples was labeled, processed and imaged in a Saphyr System and analyzed in the Bionano Access software following the manufacturer's recommendations (BionanoGenomics, San Diego CA). Results: Out of 93 pediatric AML cases included in the study, a gene fusion was identified as the primary oncogenic driver in 54 (58%) and a DNA sequence variant in 11 (12%). In 28 cases (30%), the primary genetic abnormality remained undetermined after comprehensive genetic testing. In 17/54 (31%) fusion-driven cases [17/93 (18%) overall cases], the oncogenic fusion remained undetected by karyotype analysis (cryptic). OGM was completed in 13/17 fusion positive cases with non-informative karyotype; it could not be performed in 4 cases due to lack of samples (n=2) or poor DNA quality (n=2), and is still in progress for a subset of 28 unknown cases with available material. OGM analysis successfully revealed all cryptic fusions previously detected by FISH, CMA, OncoKids® and/or RNA-Seq, including NUP98::NSD1 (n=3), KMT2A::MLLT10 (n=3), KMT2A::MLLT3 (n=2)and one case each of CBFA2T3::GLIS2, NUP98::KDM5A, DEK::NUP214, MYB::GATA1 and ETV6::MNX1. Chromosomal mechanism and structure of cryptic fusions were resolved by OGM and included: intrinsically cryptic fusions due to chromosome location of partner genes ( NUP98::NSD1, n=4; CBFA2T3::GLIS2, NUP98::KDM5A and ETV6::MNX1, n=1 each); complex rearrangements due to incompatible gene orientation ( KMT2A::MLLT10 n=2; MYB::GATA1, n=1); three-way rearrangements involving an additional chromosome ( KMT2A::MLLT3 n=2) and small insertions ( KMT2A::MLLT10 and DEK::NUP214, n=1 each). OGM also matched the performance of chromosomal microarray (CMA) analysis in detection of copy number variants. Conclusions: We confirm the significant contribution of cryptic chromosome rearrangements to pathogenesis of pediatric AML, and highlight the importance of tailored testing approaches that effectively detect highly heterogeneous fusion drivers. Our preliminary results show potential of OGM to identify clinically significant cryptic fusions in pediatric AML, and to thus markedly increase diagnostic yield compared to karyotype analysis. As a single assay that detects abnormalities currently interrogated by conventional cytogenetics, FISH and CMA, OGM may allow streamlined and more cost-effective genetic evaluation for pediatric AML.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

Pseudotyped zoonotic thogotoviruses exhibit broad entry range in mammalian cells

Viruses in the thogotovirus genus of the family Orthomyxoviridae are much less well-understood than influenza viruses despite documented zoonotic transmission and association with human disease. This study therefore developed a cell-cell fusion assay and three pseudotyping tools and used them to assess envelope function and cell tropism. Envelope glycoproteins of Dhori (DHOV), Thogoto (THOV), Bourbon, and Sinu viruses were all revealed to exhibit pH-dependent triggering of membrane fusion. Lentivirus vectors were robustly pseudotyped with these glycoproteins while influenza virus vectors showed pseudotyping compatibility, albeit at lower efficiencies. Replication-competent vesicular stomatitis virus expressing DHOV or THOV glycoproteins were also successfully generated. These pseudotyped viruses mediated entry into a wide range of mammalian cell lines, including human primary cells. The promiscuousness of these viruses suggests the use of a relatively ubiquitous receptor and their entry into numerous mammalian cells emphasize their high potential as veterinary and zoonotic diseases.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

In vitro reconstitution of herpes simplex virus 1 fusion identifies low pH as a fusion co-trigger.

HSV-1 causes lifelong, incurable infections and diseases ranging from mucocutaneous lesions to fatal encephalitis. Fusion of viral and host membranes is a critical step in HSV-1 infection of target cells that requires multiple factors on both the viral and host sides. Due to this complexity, many fundamental questions remain unanswered, such as the identity of the viral and host factors that are necessary and sufficient for HSV-1-mediated membrane fusion and the nature of the fusion trigger. Here, we developed a simplified in vitro fusion assay to examine the fusion requirements and identified low pH as a co-trigger for virus-mediated fusion in vitro. We hypothesize that low pH has a critical role in cell entry and, potentially, pathogenesis.

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several B cell malignancies and Kaposi's sarcoma. We analyzed the function of K8.1, the major antigenic component of the KSHV virion in the infection of different cells. To do this, we deleted K8.1 from the viral genome. It was found that K8.1 is critical for the infection of certain epithelial cells, e.g., a skin model cell line but not for infection of many other cells. K8.1 was found to mediate attachment of the virus to cells where it plays a role in infection. In contrast, we did not find K8.1 or a related protein from a closely related monkey virus to activate fusion of the viral and cellular membranes, at least not under the conditions tested. These findings suggest that K8.1 functions in a highly cell-specific manner during KSHV entry, playing a crucial role in the attachment of KSHV to, e.g., skin epithelial cells.

Regulation of Syntaxin3B-Mediated Membrane Fusion by T14, Munc18, and Complexin.

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV.

Cryo-EM structures of Myomaker reveal a molecular basis for myoblast fusion.

The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane protein, is essential for myoblast fusion. Here we report the cryo-EM structures of mouse Myomaker (mMymk) and Ciona robusta Myomaker (cMymk). Myomaker contains seven transmembrane helices (TMs) that adopt a G-protein-coupled receptor-like fold. TMs 2-4 form a dimeric interface, while TMs 3 and 5-7 create a lipid-binding site that holds the polar head of a phospholipid and allows the alkyl tails to insert into Myomaker. The similarity of cMymk and mMymk suggests a conserved Myomaker-mediated cell fusion mechanism across evolutionarily distant species. Functional analyses demonstrate the essentiality of the dimeric interface and the lipid-binding site for fusogenic activity, and heterologous cell-cell fusion assays show the importance of transcellular interactions of Myomaker protomers for myoblast fusion. Together, our findings provide structural and functional insights into the process of myoblast fusion.

B-237 Analytical and Clinical Performance Evaluation of the Panther Fusion® GI Parasite Assay (In Development)

Abstract Background Acute diarrhea from gastrointestinal (GI) infections is the leading cause of outpatient visits, hospitalizations, and loss of quality of life, with an estimated global impact of 500 million illnesses and 230 000 deaths annually. Most GI infections from bacteria, viruses, and parasites present similar symptoms, but successful treatment is dependent on accurate pathogen identification. For identification of parasites, microscopic testing is often used, but is laborious and often results in inconclusive or inaccurate diagnoses. Clinicians now rely on rapid and accurate molecular diagnostics to correctly identify the causative organism, which leads to optimal infection control and appropriate treatment. The Panther Fusion (PF) GI Parasite Assay is a multiplex qualitative real-time PCR assay that addresses the need for accurate and sensitive identification and detection of the most common parasites that are known to cause gastroenteritis: Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Methods Stool specimens collected in Cary-Blair (CB) are transferred to the AptimaTM Multitest collection tube (Hologic). Multitest tubes are loaded on Panther Fusion where nucleic acid isolation, amplification, and detection are performed. Analytical sensitivity was assessed using cell suspension of Cryptosporidium, Giardia or E. histolytica spiked in negative CB Stool (CBS) matrix and the limit of detection (LoD) was determined using probit. Since Cyclospora cayetanensis cannot be cultured, in-vitro transcribed RNA (IVT) was spiked in CBS matrix for LoD evaluation. Inclusivity was evaluated using cell suspensions for 20 Giardia and 15 E. histolytica strains spiked in CBS matrix. Cryptosporidium inclusivity was evaluated with synthetic nucleic acid. Specificity was evaluated by spiking cells suspensions or synthetic nucleic acid of various microbes in CBS in the presence or absence of GI targets. For clinical performance, 119 archived frozen stool specimens in CB and 40 contrived specimens were tested on Film Array GI Panel (BioFire) and PF GI Parasite Assay and results were compared. Results The LoD for Cryptosporidium, Giardia and E. histolytica was determined to be < 1 cell/mL in the Aptima Multitest tube. For C. cayetanensis, the LoD was found to be 1032 copies/mL. The PF GI Parasite Assay detected 20 strains of Giardia lamblia and 15 E. histolytica strains. In-silico and initial testing of synthetic nucleic acid demonstrated detection of 10 Cryptosporidium species. No cross-reactivity to closely related organisms or other microorganisms commonly found in the gastrointestinal tract was observed, except for cross-reactivity to Entamoeba nuttalli, which rarely infects humans. In testing of clinical remnant specimens, the PF GI Parasite Assay had a 97% PPA (35/36) for Cryptosporidium, 100% PPA (58/58) for Giardia lamblia, 100% PPA (42/42) for E. histolytica (included 40 contrived specimens) and 100% PPA (8/8) for Cyclospora with the Film Array GI Panel. Conclusion The PF GI Parasite Assay demonstrated excellent analytical and clinical performance in the detection and differentiation of Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Together with its companion Panther Fusion assays that detect bacterial and viral GI pathogens (also in development), this assay will provide a sensitive and accurate molecular tool for the automated detection of parasites in human stool.

Label-Free and Colorimetric Detection of Influenza A Virus via Receptor-Mediated Viral Fusion with Plasmonic Vesicles.

The rapid transmission and numerous re-emerging human influenza virus variants that spread via the respiratory system have led to severe global damage, emphasizing the need for detection tools that can recognize active and intact virions with infectivity. Here, this work presents a plasmonic vesicle-mediated fusogenic immunoassay (PVFIA) comprising gold nanoparticle (GNP) encapsulating fusogenic polymeric vesicles (plasmonic vesicles; PVs) for the label-free and colorimetric detection of influenza A virus (IAV). The PVFIA combines two sequential assays: a biochip-based immunoassay for target-specific capture and a PV-induced fusion assay for color change upon the IAV-PV fusion complex formation. The PVFIA demonstrates excellent specificity in capturing the target IAV, while the fusion conditions and GNP induce a significant color change, enabling visual detection. The integration of two consecutive assays results in a low detection limit (100.7919 EID50 mL-1 ) and good reliability (0.9901), indicating sensitivity that is 104.208 times higher than conventional immunoassay. Leveraging the PV viral membrane fusion activity renders the PVFIA promising for point-of-care diagnostics through colorimetric detection. The innovative approach addresses the critical need for detecting active and intact virions with infectivity, providing a valuable tool with which to combat the spread of the virus.

Membrane lytic activity of antibacterial ionenes, critical role of phosphatidylcholine (PC) and cardiolipin (CL).

Understanding the mechanism by which an antibacterial agent interacts with a model membrane provides vital information for better design of future antibiotics. In this study, we investigated two antibacterial polymers, hydrophilic C0-T-p and hydrophobic C8-T-p ionenes, known for their potent antimicrobial activity and ability to disrupt the integrity of lipid bilayers. Our hypothesize is that the composition of a lipid bilayer alters the mechanism of ionenes action, potentially providing an explanation for the observed differences in their bioactivity and selectivity. Calcein release experiments utilizing a range of liposomes to examine the impact of (i) cardiolipin (CL) to phosphatidylglycerol (PG) ratio, (ii) overall vesicle charge, and (iii) phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio on the activity of ionenes were performed. Additionally, polymer-bilayer interactions were also investigated through vesicle fusion assay and the black lipid membrane (BLM) technique The activity of C0-T-p is strongly influenced by the amount of cardiolipin, while the activity of C8-T-p primarily depends on the overall vesicle charge. Consequently, C0-T-p acts through interactions with CL, whereas C8-T-p modifies the bulk properties of the membrane in a less-specific manner. Moreover, the presence of a small amount of PC in the membrane makes the vesicle resistant to permeabilization by tested molecules. Intriguingly, more hydrophilic C0-T-p retains higher membrane activity compared to the hydrophobic C8-T-p. However, both ionenes induce vesicle fusion and increase lipid bilayer ion permeability.

Cholesterol and Ceramide Facilitate Membrane Fusion Mediated by the Fusion Peptide of the SARS-CoV-2 Spike Protein.

SARS-CoV-2 entry into host cells is mediated by the Spike (S) protein of the viral envelope. The S protein is composed of two subunits: S1 that induces binding to the host cell via its interaction with the ACE2 receptor of the cell surface and S2 that triggers fusion between viral and cellular membranes. Fusion by S2 depends on its heptad repeat domains that bring membranes close together and its fusion peptide (FP) that interacts with and perturbs the membrane structure to trigger fusion. Recent studies have suggested that cholesterol and ceramide lipids from the cell surface may facilitate SARS-CoV-2 entry into host cells, but their exact mode of action remains unknown. We have used a combination of in vitro liposome-liposome and in situ cell-cell fusion assays to study the lipid determinants of S-mediated membrane fusion. Our findings reveal that both cholesterol and ceramide lipids facilitate fusion, suggesting that targeting these lipids could be effective against SARS-CoV-2. As a proof of concept, we examined the effect of chlorpromazine (CPZ), an antipsychotic drug known to perturb membrane structure. Our results show that CPZ effectively inhibits S-mediated membrane fusion, thereby potentially impeding SARS-CoV-2 entry into the host cell.

A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

The receptor-binding domain (RBD) of spike recognizing the receptor angiotensin-converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell-vesicle content-mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell-vesicle and cell-cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike-mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike-mediated SARS-CoV-2 entry.

The Landscape of MYB/MYBL1- and Peri-MYB/MYBL1-Associated Rearrangements in Adenoid Cystic Carcinoma

Approximately 60% of adenoid cystic carcinoma (AdCC) cases are positive for MYB::NFIB or MYBL1::NFIB, whereas MYB/MYBL1 oncoprotein, a key driver of AdCC, is overexpressed in most cases. Juxtaposition of superenhancer regions in NFIB and other genes into the MYB/MYBL1 locus is an attractive oncogenic hypothesis for AdCC cases, either negative or positive for MYB/MYBL1::NFIB. However, evidence supporting this hypothesis is insufficient. We examined 160 salivary AdCC cases for rearrangements in MYB/MYBL1 loci and peri-MYB/MYBL1 areas (centromeric and telomeric areas of 10 Mb each) using formalin-fixed, paraffin-embedded tumor sections. For the detection of the rearrangements, we employed conventional fluorescence in situ hybridization split and fusion assays and a 5 Mb fluorescence in situ hybridization split assay. The latter is a novel assay that enabled us to detect any possible splits within a 5 Mb distance of a chromosome. We found MYB/MYBL1- and peri-MYB/MYBL1-associated rearrangements in 149/160 patients (93%). AdCC cases positive for rearrangements in MYB, MYBL1, the peri-MYB area, and the peri-MYBL1 area numbered 105 (66%), 20 (13%), 19 (12%), and 5 (3%), respectively. In 24 peri-MYB/MYBL1 rearrangement-positive cases, 14 (58%) were found to have a juxtaposition of the NFIB or RAD51B locus into the MYB/MYBL1 loci. On comparing with a tumor group positive for MYB::NFIB, a hallmark of AdCC, other genetically classified tumor groups had similar features of overexpression of the MYB transcript and MYB oncoprotein as detected by semiquantitative RT-qPCR and immunohistochemistry, respectively. In addition, clinicopathological and prognostic features were similar among these groups. Our study suggests that peri-MYB/MYBL1 rearrangements may be a frequent event in AdCC and may result in biological and clinicopathological consequences comparable to MYB/MYBL1 rearrangements. The landscape of MYB/MYBL1 and peri-MYB/MYBL1 rearrangements shown here strongly suggests that juxtaposition of superenhancers into MYB/MYBL1 or peri-MYB/MYBL1 loci is an alteration that acts as a key driver for AdCC oncogenesis and may unify MYB/MYBL1 rearrangement-positive and negative cases.

Verifying AXL and putative proteins as SARS-CoV-2 receptors by DnaE intein-based rapid cell-cell fusion assay.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

Detection of salivary gland and sinonasal fusions by a next-generation sequencing based, ligation-dependent, multiplex RT-PCR assay.

The discovery of tumour type-specific gene fusion oncogenes in benign and malignant salivary gland and sinonasal (SGSN) tumours has significantly increased our knowledge about their molecular pathology and classification. We developed a new targeted multiplexed next-generation sequencing (NGS)-based method that utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR) to detect oncogenic fusion transcripts involving 116 genes, leading to 96 gene fusions known to be recurrently rearranged in these tumours. In all, 180 SGSN tumours (formalin-fixed, paraffin-embedded samples, 141 specimens and 39 core needle biopsies) from the REFCORpath (French network for rare head and neck cancers) with previously identified fusion genes by fluorescent in situ hybridisation (FISH), RT-PCR, or molecular immunohistochemistry were selected to test its specificity and sensitivity and validate its diagnostic use. Tested tumours encompassed 14 major tumours types, including secretory carcinoma, mucoepidermoid carcinoma, adenoid cystic carcinoma, salivary gland intraductal carcinoma, clear cell carcinoma, pleomorphic adenoma, adamantinoma-like Ewing Sarcoma, EWSR1::COLCA2 sinonasal sarcoma, DEK::AFF2 sinonasal carcinoma, and biphenotypic sinonasal sarcoma. In-frame fusion transcripts were detected in 97.8% of cases (176/180). Gene fusion assay results correlated with conventional techniques (immunohistochemistry [IHC], FISH, and RT-PCR) in 176/180 tumours (97.8%). This targeted multiplexed NGS-based LD-RT-PCR method is a robust, highly sensitive method for the detection of recurrent gene fusions from routine clinical SGSN tumours. It can be easily customized to cover new fusions. These results are promising for implementing an integrated NGS system to rapidlydetect genetic aberrations, facilitating accurate, genomics-based diagnoses, and accelerate time to precision therapies in SGSN tumours.

β-Cyclodextrins as affordable antivirals to treat coronavirus infection

The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested their efficacy as antivirals against α and β coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four drugs, OSW-1, U18666A, hydroxypropyl-β-cyclodextrin (HβCD) and phytol, showed in vitro antiviral activity against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HβCD and U18666A, yet only HβCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, β-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. β-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to β-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of β-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals.