Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies.

IMPORTANCE: The wide endemic range of mosquito-vectored flaviviruses-such as Zika virus and dengue virus serotypes 1-4-places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue toward development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses. Here, we measure how single amino acid mutations in viral envelope protein affect neutralizing antibodies with both broad and narrow specificities. We find that broadly neutralizing antibodies with potential as vaccine prototypes or biological therapeutics are quantifiably more difficult to escape than narrow, virus-specific neutralizing antibodies.

Canine Oral Lymphoma: A Review and 3 Case Studies.

Canine oral lymphoma is an infrequently diagnosed malignancy in dogs and reports in the literature are lacking. Most cases are due to epitheliotropic lymphoma, in which lesions are often multifocal and involve the skin as well as the oral cavity. Epitheliotropic lymphoma is an uncommon form of lymphoma that is characterized by neoplastic T-lymphocyte tropism for epithelial tissues. However, not all cases of oral lymphoma are due to epitheliotropic lymphoma, and B-cell disease is also possible. This article describes very different cases of oral lymphoma in dogs including the history at presentation, examination and radiographic findings, treatments, and outcomes. The cases highlight the variability in signalment, examination findings, and clinical courses that are found with oral lymphoma.

Trainee Effect on Procedural Efficiency is Limited in Vascular Surgery Operations.

BACKGROUND: Surgical residents prepare during their training for independent operating experience. However, there is a fine balance between supervised intraoperative teaching and the need to keep operations short since this is associated with improved patient safety. We aim to understand if the composition of the vascular surgical team-presence of anesthesia and surgical trainees as well as the number of circulating nurses-affects elective operative times at our institution. As a secondary aim, we analyzed how time of day affects overall operative time. METHODS: We performed a retrospective review of all vascular surgery elective operations occurring between January 1, 2019, and October 15, 2021. Our reference operation between procedures was the construction of an arteriovenous fistula (AVF). Reference teams included circulating staff (fewer than two nurses), anesthesia (anesthesiologist with certified registered nurse anesthetist [CRNA]), and surgery (surgeon with nurse practitioner). The primary dependent variable was the time interval in minutes from wheels-in to wheels-out of surgery, which was divided into three subintervals: wheels-in to cut, cut to close, and close to wheels-out. Univariate analysis was performed to examine each surgical procedure's distribution of wheels-in to wheels-out time interval. Linear regression was performed to determine the effect of team composition and time of day on operative durations. RESULTS: We included a total of 853 vascular operations. Regarding overall operative time, different procedures took various amounts of time compared with the reference operation (AVF creation). Amputations and arteriograms were shorter (-30 min, P = 0.03, and -12 min, P = 0.05, respectively). Other procedures were longer: endarterectomy (+48 min, P < 0.01), rib resection (+78 min, P < 0.01), endovascular aorta repair (+120 min, P < 0.01), lower extremity bypass (+170 min, P < 0.01), and open aortic repair (+410 min, P < 0.01). No significant difference was found in carotid artery stent placement. Overall, there was a significant reduction in the close to wheels-out interval for anesthesiologists with a trainee (mean: -2.4 min; 95%; CI: -4.7, -0.12; P = 0.04). AVF took significantly more time with a surgical resident: wheels-in to cut time (mean: +4.2 min; 95%; CI: 0.92, 7.4; P = 0.01) and cut to close time (mean: +13 min; 3.2, 23; P < 0.01). Arteriogram wheels-in to cut time took longer with a surgeon alone (mean: +5.6; 95%; CI: 0.29, 11; P = 0.04). There were no other statistically significant findings with change in composition of the surgical team or changes in start time. CONCLUSIONS: General surgery residents generally do not add time to vascular surgery cases but may do so in certain cases, perhaps when they are given more autonomy (i.e. AVF creation). Future studies should look at multiple centers, specific vascular procedures, and level of training to explore whether experience among residents (i.e., intern versus senior resident) and case complexity play a role in procedural length, as this may indirectly affect attending surgeon burnout and patient outcomes.

Optimising tissue acquisition and the molecular testing pathway for patients with non-small cell lung cancer: A UK expert consensus statement.

Targeted therapy against actionable variants has revolutionised the treatment landscape for non-small cell lung cancer (NSCLC). Approximately half of NSCLC adenocarcinomas have an actionable variant, making molecular testing a critical component of the diagnostic process to personalise therapeutic options, optimise clinical outcomes and minimise toxicity. Recently, genomic testing in England has undergone major changes with the introduction of Genomic Laboratory Hubs, designed to consolidate and enhance existing laboratory provision and deliver genomic testing as outlined in the National Genomic Test Directory. Similar changes are ongoing in Scotland, Wales and Northern Ireland. However, multiple challenges exist with current tissue acquisition procedures and the molecular testing pathway in the UK, including quantity and quality of available tissue, adequacy rates, test availability among genomic laboratories, turnaround times, multidisciplinary team communication, and limited guidance and standardisation. The COVID-19 pandemic has added an extra layer of complexity. Herein, we summarise best practice recommendations, based on expert opinion, to overcome existing challenges in the UK. The least invasive biopsy technique should be undertaken with the aim of acquiring the greatest quality and quantity of tissue. Use of sedation should be considered to improve patient experience. Rapid on-site evaluation may also be useful to help guide adequate sampling, and liquid biopsy may be beneficial in some instances. Sample processing should be appropriate to facilitate biomarker testing, in particular, next-generation sequencing for comprehensive genomic information. Steps to optimise tissue utilisation and turnaround times, such as planning of tissue usage, limiting immunohistochemistry, tumour enrichment, and reflex testing at diagnosis, should be implemented. Guidelines for tissue acquisition and sample processing may help to improve sample adequacy to perform downstream testing. Communication among genomic laboratories will help to standardise test availability across England and local auditing could identify further areas for optimisation, including ways to improve turnaround times and adequacy rates.

Vulval extra-mammary Paget's disease: the master of disguise revealed.

Extra-mammary Paget's disease is a rare cancer affecting the anogenital region and can present with a myriad of symptoms. In women, the diagnosis of Paget's disease can be challenging as it mimics several other vulval conditions. It is important to promptly recognise this disease because of its potential association with synchronous tumours, such as colorectal adenocarcinoma. The mainstay of treatment is with immunomodulating therapies or surgery, but unfortunately the risk of recurrence is high and appears to be independent of treatment choice and tissue margin status. There is growing evidence to move away from traditional surgical excision to using topical therapy, such as imiquimod, as surgery can often be extensive and disfiguring with a prolonged recovery time. There is considerable psychosexual and physical morbidity associated with Paget's disease, largely owing to multiple surgical recurrences. As recurrences can occur several years after the initial presentation, long-term follow up of patients is recommended.

Rubidium and caesium aluminyls: synthesis, structures and reactivity in C-H bond activation of benzene.

Expanding knowledge of low valent aluminium chemistry, rubidium and caesium aluminyls are reported to complete the group 1 (Li-Cs) set of metal aluminyls. Both compounds crystallize as a contacted dimeric pair supported by M⋯π(arene) interactions with a pronounced twist between aluminyl units. Density functional theory calculations show symmetrical bonding between the M and Al atoms, with an Al centred lone-pair donating into vacant Rb and Cs orbitals. Interestingly, despite their structural similarity the Cs aluminyl enables C-H bond activation of benzene, but not the Rb aluminyl reflecting the importance of the alkali metal in these heterobimetallic systems.

Case Report: Multiple colorectal cancers in a patient with Ulcerative colitis and Lynch syndrome: Is there a role for prophylactic colectomy? A short report and review of literature.

It is a known fact that Lynch syndrome (LS) and Ulcerative colitis (UC) are individually associated with increased risk of colorectal cancer. While there is no conclusive evidence to demonstrate a cumulative risk when these two conditions coexist, available data suggest early onset and synchronous cancers are synonymous to this group. We have reported an unusual case of multiple synchronous colorectal cancers in a young man with ulcerative colitis and Lynch syndrome also known as Hereditary Nonpolyposis Colorectal Cancer (HNPCC) gene mutation. We propose that conducting a detailed genetic mutation profile in LS patients may play a key role in guiding the intensity of endoscopic surveillance and that a concerted, pragmatic, patient guided approach should be adopted on the subject of prophylactic colectomy when UC and LS co-exist.

Glenoid vault perforation in total shoulder arthroplasty: Do we need computer guidance?

The proliferation of computer 3D simulation and computer-generated guides is aimed at minimizing perforation of the glenoid vault by glenoid pegs in shoulder arthroplasty, based on assumptions that perforation leads to worse outcomes by component loosening and potential failure. We evaluated outcomes of glenoid peg perforation testing the assumption that perforation produces worse results. Eighty-three shoulders underwent shoulder arthroplasty with pegged hybrid fixation (bone-ingrowth flanged central glenoid peg and peripheral cemented pegs) without precision signal injector guides or use of 3D planning software. Outcomes were determined by American Shoulder and Elbow Score and Oxford Shoulder Score. Fine slice CT determined the presence of vault perforation and the extent of lucent lines at the prosthesis-bone interface and bony morphology of the vault perforation. Follow-up was 46.7 months (24-99). Seven shoulders (8%) demonstrated perforation of glenoid vault. Bony ingrowth and cortical overgrowth occurred despite perforation, with no clinically significant differences in clinical or radiological outcomes in shoulders with and without glenoid vault perforation. None of these patients underwent revision surgery. Despite not utilizing computer planning and/or guides, 92% of implants did not perforate the glenoid vault. However, glenoid vault perforation in our series produced excellent outcomes with no increased risk of revision as a result of glenoid vault perforation.

Medium-Term Clinical and Radiographic Results of an All-Polyethylene, Pegged, Bone-Ingrowth Glenoid Component: A Concise Follow-up of a Previous Report.

ABSTRACT: We previously reported the mean 4-year outcomes of anatomic total shoulder replacement using an all-polyethylene, pegged, hybrid-fixation (bone ingrowth and cement) glenoid component. In the present study, we report on that patient cohort after another 4 years of follow-up (mean, 101 months; range, 77 to 146 months). At that time, the median American Shoulder and Elbow Surgeons (ASES) score was 92 points (interquartile range [IQR], 81.7 to 98.3) and the median Oxford Shoulder Score was 47 points (IQR, 41 to 48). Osseointegration, demonstrated by bone ingrowth between the flanges on the central peg as seen on coronal computed tomography (CT), was complete in 75% of the shoulders, partial in 21%, and absent in 4%. There were radiolucent lines at the bone-prosthesis interface on CT, with a median Yian score of 1 (IQR, 0 to 2; range, 0 to 18). The conclusion in the present study was that shoulder arthroplasty with an all-polyethylene, hybrid-fixation (bone ingrowth and cement) pegged glenoid component has durable clinical and radiographic outcomes at medium-term follow-up. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion.

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

Pelvic and Ovarian Recurrence of Small HPV-associated Cervical Adenocarcinoma With Transformation to Neuroendocrine Carcinoma.

The phenomenon of small human papillomavirus-associated cervical adenocarcinomas involving the ovary via a transuterine and transtubal route is uncommon but well described in the literature. We report a unique case of a small human papillomavirus-associated cervical adenocarcinoma spreading to both ovaries and the pelvis via this route 22 mo after loop excision and trachelectomy and developing into a high-grade neuroendocrine carcinoma in the metastasis.

Chronic inflammation and other changes are significant components of clinically fibrotic strictures in Crohn's disease: a histological study of resected strictures clinically characterized as noninflamed.

BACKGROUND: Strictures related to Crohn's disease due to fibrosis are a result of an exaggerated tissue remodelling response to inflammation, characterized by accumulation of collagen-rich extracellular matrix produced by mesenchymal cells. OBJECTIVES: The objective of this study was to characterize histological changes seen in resected 'fibrotic' strictures to better understand individual components of intestinal stenosis. METHODS: We identified patients undergoing surgery for ileal Crohn's disease secondary to symptomatic stricturing disease (Montreal B2) using the histopathology database at Queen Elizabeth Hospital in Birmingham, UK, between 2012 and 2017. Phenotypic data were recorded and resection specimens reviewed. Two independent pathologists applied the semiquantitative scoring system previously developed by us to the microscopic images. Data were analyzed using the possible maximum total score (%PMTS). RESULTS: Forty-eight patients (M = 25) were included. with median disease duration of 7 years (range 0.25-39 years); nearly two-thirds had ileocolonic distribution (L3). In this cohort, despite presurgery diagnosis of noninflamed fibrosis, chronic inflammation was noted to be a prominent component of all strictures. The histological scoring showed presence of several other prominent findings such as muscular hyperplasia and volume expansion.There was statistically significant positive correlation between chronic inflammation and fibrosis and muscular hyperplasia. CONCLUSION: The histological features of Crohn's disease-related strictures show multiple changes in multiple layers and not simply fibrosis. In our cohort, despite the observation prior to surgery that strictures were clinically considered fibrotic, the finding of chronic inflammation as a dominant component at a histological level in the resection is important. The findings might suggest that one of the main drivers of progressive fibrosis is the inflammatory component, which probably is never fully resolved.

Convergent epicardial-endocardial ablation for treatment of long-standing persistent atrial fibrillation: A review of literature.

BACKGROUND AND AIM OF STUDY: The convergent procedure (CVP) is a hybrid ablation technique via a subxiphoid incision that has recently emerged as a treatment option for non-paroxysmal atrial fibrillation (npAF). By combining endocardial and epicardial ablation into a simultaneous or staged procedure, the pulmonary vein and posterior left atrium can be isolated with transmural lesion sets while minimizing the risk of proarrhythmic gaps that are a known limitation with endocardial linear lesion sets. We reviewed the 12-month outcomes in patients who underwent CVP compared to those who underwent endocardial catheter ablation (CA) and surgical ablation (SA). METHODS: A literature search was conducted using the PubMed database for publications related to CVP. Selected studies included detailed 12-month follow-up of patients, patient characteristics, periprocedural complications, use of antiarrhythmic drugs (AADs), and monitoring method. RESULTS: Five studies with 340 patients who underwent CVP between January 2009 and March 2017 were selected for this review. A total of 8.5% of patients had paroxysmal AF (pAF), 42.2% had persistent AF (peAF), and 49.1% had long-standing persistent AF (lspAF). At 12 months, 81.9% of patients were in sinus rhythm, while 54.1% of patients were in sinus rhythm while not taking AADs. The overall complication rate was 10%. CONCLUSION: CVP had better 1-year efficacy in eliminating AF when compared to CA. However, SA, specifically the Cox Maze IV, had lower rates of AF recurrence in the npAF patient population. Despite its promising 1-year efficacy rates, the periprocedural complication rate for CVP was significantly higher than both CA and SA.

The Clinicopathological and Molecular Associations of PD-L1 Expression in Non-small Cell Lung Cancer: Analysis of a Series of 10,005 Cases Tested with the 22C3 Assay.

PD-L1 expression testing is mandatory prior to pembrolizumab prescription in non-small cell lung cancer. Our service offers PD-L1 testing using the PD-L1 IHC 22C3 pharmDx assay, in parallel with EGFR, ALK, ROS1 and (in some cases) KRAS testing. We correlate PD-L1 expression in 10,005 tumours with patient age and sex, with tumour histological subtypes, with the sampling modality and type of tissue, and with the presence of other molecular alterations. PD-L1 expression testing was performed using the aforementioned assay; tumour proportion scores (TPS) of 1 and 50% were taken as cut-offs for low and high positivity, respectively. EGFR testing was performed using the cobas® EGFR Mutation Test v2. ALK testing was performed using the VENTANA ALK (D5F3) CDx Assay. KRAS testing was performed using pyrosequencing. TPS <1% was seen in 44.4% of tumours, 1-49% in 25.0% and ≥ 50% in 30.6%. We identified no significant relationship with age. Female patients were slightly more likely to express PD-L1. Poorly-differentiated tumour histology and ALK translocation were significantly associated with PD-L1 expression. Rare EGFR mutations tended to be associated with PD-L1 expression. Pleural and nodal metastases were more likely to express PD-L1 than primary tumours, but biopsy and cytological specimens did not show different PD-L1 expression rates. Our data show that the means of acquiring a tumour sample (biopsy versus cytology) does not have a significant impact on PD-L1 expression. However, we found that certain metastatic sites were associated with significantly higher expression rates, which has substantial implications for selection of tissue for testing.

Deep Mutational Scanning Comprehensively Maps How Zika Envelope Protein Mutations Affect Viral Growth and Antibody Escape.

Functional constraints on viral proteins are often assessed by examining sequence conservation among natural strains, but this approach is relatively ineffective for Zika virus because all known sequences are highly similar. Here, we take an alternative approach to map functional constraints on Zika virus's envelope (E) protein by using deep mutational scanning to measure how all amino acid mutations to the E protein affect viral growth in cell culture. The resulting sequence-function map is consistent with existing knowledge about E protein structure and function but also provides insight into mutation-level constraints in many regions of the protein that have not been well characterized in prior functional work. In addition, we extend our approach to completely map how mutations affect viral neutralization by two monoclonal antibodies, thereby precisely defining their functional epitopes. Overall, our study provides a valuable resource for understanding the effects of mutations to this important viral protein and also offers a roadmap for future work to map functional and antigenic selection to Zika virus at high resolution.IMPORTANCE Zika virus has recently been shown to be associated with severe birth defects. The virus's E protein mediates its ability to infect cells and is also the primary target of the antibodies that are elicited by natural infection and vaccines that are being developed against the virus. Therefore, determining the effects of mutations to this protein is important for understanding its function, its susceptibility to vaccine-mediated immunity, and its potential for future evolution. We completely mapped how amino acid mutations to the E protein affected the virus's ability to grow in cells in the laboratory and escape from several antibodies. The resulting maps relate changes in the E protein's sequence to changes in viral function and therefore provide a valuable complement to existing maps of the physical structure of the protein.

An Influenza Virus Entry Inhibitor Targets Class II PI3 Kinase and Synergizes with Oseltamivir.

Two classes of antivirals targeting the viral neuraminidase (NA) and endonuclease are currently the only clinically useful drugs for the treatment of influenza. However, resistance to both antivirals has been observed in clinical isolates, and there was widespread resistance to oseltamivir (an NA inhibitor) among H1N1 viruses prior to 2009. This potential for resistance and lack of diversity for antiviral targets highlights the need for new influenza antivirals with a higher barrier to resistance. In this study, we identified an antiviral compound, M85, that targets host kinases, epidermal growth factor receptor (EGFR), and phosphoinositide 3 class II ß (PIK3C2ß) and is not susceptible to resistance by viral mutations. M85 blocks endocytosis of influenza viruses and inhibits a broad-spectrum of viruses with minimal cytotoxicity. In vitro, we found that combinations of M85 and oseltamivir have strong synergism. In the mouse model for influenza, treatment with the combination therapy was more protective against a lethal viral challenge than oseltamivir alone, indicating that development of M85 could lead to combination therapies for influenza. Finally, through this discovery of M85 and its antiviral mechanism, we present the first description of PIK3C2ß as a necessary host factor for influenza virus entry.

Human Monoclonal Antibodies Potently Neutralize Zika Virus and Select for Escape Mutations on the Lateral Ridge of the Envelope Protein.

The mosquito-borne Zika virus (ZIKV) has been causing epidemic outbreaks on a global scale. Virus infection can result in severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we characterized monoclonal antibodies isolated from a patient with an active Zika virus infection that potently neutralized virus infection in Vero cells at the nanogram-per-milliliter range. In addition, these antibodies enhanced internalization of virions into human leukemia K562 cells in vitro, indicating their possible ability to cause antibody-dependent enhancement of disease. Escape variants of the ZIKV MR766 strain to a potently neutralizing antibody, AC10, exhibited an amino acid substitution at residue S368 in the lateral ridge region of the envelope protein. Analysis of publicly availably ZIKV sequences revealed the S368 site to be conserved among the vast majority (97.6%) of circulating strains. We validated the importance of this residue by engineering a recombinant virus with an S368R point mutation that was unable to be fully neutralized by AC10. Four out of the 12 monoclonal antibodies tested were also unable to neutralize the virus with the S368R mutation, suggesting this region to be an important immunogenic epitope during human infection. Last, a time-of-addition infection assay further validated the escape variant and showed that all monoclonal antibodies inhibited virus binding to the cell surface. Thus, the present study demonstrates that the lateral ridge region of the envelope protein is likely an immunodominant, neutralizing epitope.IMPORTANCE Zika virus (ZIKV) is a global health threat causing severe disease in humans, including microcephaly in newborns and Guillain-Barré syndrome in adults. Here, we analyzed the human monoclonal antibody response to acute ZIKV infection and found that neutralizing antibodies could not elicit Fc-mediated immune effector functions but could potentiate antibody-dependent enhancement of disease. We further identified critical epitopes involved with neutralization by generating and characterizing escape variants by whole-genome sequencing. We demonstrate that the lateral ridge region, particularly the S368 amino acid site, is critical for neutralization by domain III-specific antibodies.

Zika Virus Subverts Stress Granules To Promote and Restrict Viral Gene Expression.

Flaviviruses limit the cell stress response by preventing the formation of stress granules (SGs) and modulate viral gene expression by subverting different proteins involved in the stress granule pathway. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity, of stress granule proteins. We also investigated the role of different stress granule proteins in ZIKV infection by using target-specific short interfering RNAs to deplete Ataxin2, G3BP1, HuR, TIA-1, TIAR, and YB1. Knockdown of TIA-1 and TIAR affected ZIKV protein and RNA levels but not viral titers. Conversely, depletion of Ataxin2 and YB1 decreased virion production despite having only a small effect on ZIKV protein expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia Luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, where G3BP1 functions to promote ZIKV infection while HuR exhibits an antiviral effect. The results of ZIKV relocalizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis and contribute to cellular gene dysregulation and ZIKV pathogenesis.IMPORTANCE Many viruses inhibit SGs. In this study, we observed that ZIKV restricts SG assembly, likely by relocalizing and subverting specific SG proteins to modulate ZIKV replication. This ZIKV-SG protein interaction is interesting, as many SG proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different SG proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of ZIKV modulating SG assembly and subverting specific SG proteins are alterations to cellular mRNA transcription, splicing, stability, and translation. Such changes in cellular ribostasis could profoundly affect neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine ZIKV infection. Our study provides new insights into virus-host interactions and the identification of the SG proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.

Large-Scale EGFR Mutation Testing in Clinical Practice: Analysis of a Series of 18,920 Non-Small Cell Lung Cancer Cases.

We make use of a very large dataset of non-small cell lung cancer specimens to examine the molecular epidemiology of EGFR mutations, particularly with respect to rare and compound mutations, and to non-adenocarcinoma histological subtypes. We also demonstrate the feasibility of large-scale EGFR mutation screening using the full range of specimens encountered in routine practice. We retrospectively reviewed 18,920 unselected EGFR mutation results from our centre between July 2009 and October 2016, using Qiagen's therascreen EGFR RGQ PCR Kit. Mutation rates were correlated with patient demographics and tumour histology. Our testing success rate was 93.9%, with similar success rates using histological and cytological specimens. Rare, potentially-targetable mutations accounted for 9.5% of all mutations detected. We identified a 2.5% mutation rate in tumours diagnosed as squamous cell carcinomas. There was a trend towards increasing EGFR mutation rates with increasing age, and while Del19 was the commonest mutation in the young, L858R predominated in the elderly. We found that EGFR mutation heterogeneity is rare within tumours and between primary and metastatic deposits. Our data demonstrate that large-scale, reflex EGFR mutation testing is feasible and affordable in the context of a publicly-funded health system. Furthermore, we have shown that the use of techniques sensitive only to classical mutations and selection of patients on the grounds of age, sex and histology denies patients access to potentially beneficial TKI therapy.