Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalisation via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist which counter glioblastoma invasion. Here, we report that inhibition of Ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR over-expression using timelapse microscopy, two orthotopic glioblastoma models and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high throughput, super resolution imaging and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets whilst histological, protein expression and spatial transcriptomics using glioblastoma tumour specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalisation of integrins at growth cone-like structures, resulting in a tumour microtube retraction defect. The biological relevance and translational potential of these findings was confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSION: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far reaching clinical benefits beyond radiosensitisation.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Allometric scaling of flow-mediated dilation: is it always helpful?

Flow-mediated dilation (FMD) is calculated as the greatest percent change in arterial diameter following an ischaemic challenge. This Traditional %FMD calculation is thought to have statistical bias towards baseline diameter (Dbase ), which is reduced by allometric scaling. This study examined whether allometric scaling FMD influenced the difference between a group of healthy young and older adults compared to the Traditional %FMD, and to determine whether a New (allometric) scaling %FMD improved the ability to obtain individually scaled FMD. Popliteal artery FMD was assessed in 18 young (26 ± 3 years) and 17 older adults (77 ± 5 years). 'Corrected' mean FMD was generated from a log-linked ANCOVA model. Individual %FMD was evaluated using three calculations: (1) Traditional %FMD calculation; (2) Atkinson (allometric) scaling %FMD (peak diameter (Dpeak)/(Dbasescalingexponent)); and (3) New scaling %FMD ((Dpeak-Dbase)/(Dbasescalingexponent)). Traditional %FMD was significantly larger in young (5·82 ± 2·58%) versus old (3·72 ± 1·26%). 'Corrected' FMD means (Y: 5·97 ± 2·12%; O: 3·98 ± 2·06%) were similar to Traditional %FMD; however, the logarithmic transformation prevents statistical interpretation of group differences. Individually scaled %FMD using the Atkinson scaling resulted in values that were corrected for variations in Dbase but that were twofold to threefold larger than those of the Traditional calculation. New scaling %FMD resulted in values that were similar to values expected (Y: 6·21 ± 2·75%; O: 3·98 ± 1·36%); however, it did not effectively correct for variation in Dbase . Recommendations regarding the advantages of allometrically scaling %FMD should be made with caution until research clearly establishes the benefits of this approach.

The DNA sequence and biological annotation of human chromosome 1.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

Long-term clinico-radiological assessment of endoscopic stapling of pharyngeal pouch: a series of cases.

The endoscopic division of the pharyngeal pouch wall with a mechanical stapling device has become increasingly popular. When compared to open excision, the reduced operative time, early resumption of oral intake, and short in-patient stay with no early recurrence of symptoms, are the proposed advantages. Small pouches or thick walled pouches are not suitable for stapling. From December 1996 to December 1999, 32 patients were admitted to the Aberdeen Royal Infirmary for endoscopic stapling of a pharyngeal pouch. Five patients were unsuitable for stapling. In addition, three patients were treated for pouch recurrence after an external approach. Two patients required repeat stapling at a later date. Our results are encouraging in terms of short operation time and hospital admission, improvement of symptoms and minimal complication rate. Fifteen patients were assessed 24 months after the procedure with satisfaction surveys and contrast swallow X-rays. Subjective improvement was sustained throughout this period, despite radiological evidence of persistence of the pharyngeal pouch.

Necrotizing sarcoid granulomatosis with extrapulmonary involvement.

Pulmonary lesions, with sarcoid-like granulomas exhibiting noncaseous necrosis, with associated granulomatous arteritis fulfil the diagnostic criteria of necrotizing sarcoid granulomatosis (NSG). We report the case of a woman who presented with recurrent headaches, transient right hemipareses and left-sided ophthalmoplegia. An excised left retro-orbital lesion demonstrated sarcoid like changes, and the illness responded to steroid therapy. Twelve years later, the patient developed a tumour in the right lung. The resected specimen showed the histological hallmarks of NSG, and careful review of the retro-orbital lesion, removed 12 years previously, revealed similar histology. Extrapulmonary involvement in NSG is rare and has been histologically proven on only one previous occasion. The presentation of necrotizing sarcoid granulomatosis in two different systems 12 years apart is unusual and was considered worth reporting.

The absence of Harvey ras mutations during development and progression of squamous cell carcinomas of the head and neck.

We have examined the incidence of Harvey ras mutations in human squamous cell carcinomas (SCC) of the upper aerodigestive tract using the polymerase chain reaction (PCR) followed by direct sequencing. No mutations were detected at codons 12, 13, 59 or 61 of this gene in any of six papillomas, five erythroplakias, 56 squamous cell carcinomas, and 16 SCC cell lines. Some of the SCC were lymph node metastases (three) or tumours which had recurred following radiotherapy (seven). We conclude that Harvey ras mutations are not a common event in the pathogenesis or recurrence of SCCs from Caucasian subjects, in contrast to the situation with Indian populations (Saranath et al., 1991).

Magnetic resonance imaging of the parotid glands using inversion-recovery sequences at 0.08 T.

One hundred and eight examinations on 103 patients with suspected disease of the salivary glands were studied using a 0.08 T resistive magnet and inversion-recovery pulse sequences. Sixty-eight patients who had a mass lesion within a salivary gland later had surgery, and specimens were obtained for histological diagnosis. The remaining 35 patients were diagnosed on clinical grounds. In all cases the clinical findings were compared with the appearances on magnetic resonance imaging (MRI). The use of inversion-recovery pulse sequences allowed accurate localization of all tumour masses and, because of the clarity with which blood vessels were displayed, the precise relationship of any parotid mass to the retromandibular vessels and hence the facial nerve was possible. Whilst MRI did not display any pathognomonic features to allow the differentiation of malignant cell types or the differentiation of invasive malignant tumours from chronic inflammatory disease, it was possible to differentiate parotitis from Mikulicz's disease and to diagnose cysts and pleomorphic adenomas by their appearances on MRI.

Ectopic thyroid malignancy in the midline of the neck (a case report and literature review).

A 71-year-old female presented with a midline neck mass. The clinical impression was that of a thyroglossal cyst but preoperative assessment suggested a solid lesion, possibly malignant. The mass was removed surgically using the Sistrunk technique and shown pathologically to be a mixed papillary-follicular carcinoma of the thyroid with no cystic elements. Clinically and radiologically the thyroid was normal and thyroidectomy was not performed. This management is discussed along with a brief review of the relevant literature on the pathology and treatment of similar lesions.

Pharyngeal dermoid leading to respiratory failure in a neonate.

Difficulty in resuscitating a neonate using bag and mask ventilation was followed by later episodes of apnoea and cyanosis. Intubation relieved the respiratory failure and revealed a large fleshy polyp obstructing the posterior pharynx. Computerised tomography showed there was no superior extension of the lesion and the polyp was removed completely on operation. Histological examination showed this lesion to be a dermoid or "Hairy Polyp".

Carcinoid tumour of the larynx. A report of two cases.

Two cases of carcinoid tumours of the larynx are described. One was histologically a typical carcinoid whilst the other showed cellular atypia, numerous mitoses and invasion of the surrounding tissues. Both were argyrophilic but not argentaffin positive whilst electron microscopy demonstrated the presence of neurosecretory granules and amyloid. The previous literature on these rare tumours of the larynx is reviewed.