SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection.

Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.

Aquaporin-4 expression is increased in edematous meningiomas.

Peritumoral edema is frequently present in meningiomas and can result in serious morbidity and mortality. The aquaporins (AQPs) are a family of membrane protein water channels with an integral role in water transport and maintenance of fluid balance. AQP4, increased in edematous human brain tumors such as astrocytomas and metastases, is present in the astrocytic foot processes adjacent to endothelial cells and may therefore have a role in cerebral edema formation. The objective of this study is to investigate the expression of AQP4 in meningiomas and to correlate their expression with peritumoral edema. Fresh human meningioma specimens (17) were obtained and immunohistochemical staining and Western blot analysis was performed for AQP4. The peritumoral edema index (EI) was calculated based on MRI post-processed to calculate the tumor and edema volume. Overexpression of AQP4 was associated with significant peritumoral edema. Immunohistochemistry showed upregulation of AQP4 throughout the specimens. Therefore, we conclude that increased expression of AQP4 is associated with peritumoral edema in meningiomas. This suggests that AQP4 overexpression can lead to abnormal water transport and edema formation in meningiomas. The inhibition of AQP4 water channels is a potential therapeutic option to reduce the adverse effects of peritumoral edema in meningiomas.

Combinatorial-approached neuroprotection using pan-caspase inhibitor and poly (ADP-ribose) polymerase (PARP) inhibitor following experimental stroke in rats; is there additional benefit?

Energy requiring apoptosis and presumably unregulated necrosis are considered conceptually and morphologically distinct forms of cell death which have been initially identified as two exclusive pathways. However, several apoptotic characteristics have been observed in the necrotic core lesion in ischemia which led to the controversial theory that cell death advances via a number of hybrid pathways among a continuum between the two processes. ATP availability has been shown to influence the decision between apoptosis and necrosis. The aims of our study are 1) to determine if combined inhibitors administration of pan-caspase inhibitor Carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and non-selective poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) can further reduce infarct volume compared to single modality of either inhibitor following ischemic insult, 2) to ascertain the pharmacological intervention up to 24 hour post-middle cerebral artery occlusion (MCAo), and 3) to correlate intracellular ATP level with infarct volume. Single modality treatment was optimised at 3 mg/kg z-VAD-fmk and 30 mg/kg 3-AB with infarct volume measured at 24.13%+/-3.89% and 26.98%+/-2.22% respectively, while untreated control group was determined at 45.97%+/-1.86%. Combined inhibitors treatment rendered further reduction in infarct volume, measuring 7.228%+/-1.988%, 21.02%+/-1.06%, 24.40%+/-2.12% at 30 min, 6 h, 24 h post-ischemia respectively. In conclusion, the combined inhibitors administration of both z-VAD-fmk and 3-AB show further increased in infarct volume reduction with our ischemic model up to the 24 hour post-MCAo. However, in our in vivo study, no correlation between intracellular ATP level and infarct size was established.

Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy.

Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs). Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed. CTCs were captured by microsieve technology and enumerated, while three different methods of EBV cfDNA quantification were applied, including an in-house qPCR assay for BamHI-W fragment, a CE-IVD qPCR assay (Sentosa ®) and a dPCR (Clarity™) assay for Epstein-Barr nuclear antigen 1 (EBNA1). EBV cfDNA quantitation by all workflows showed stronger correlation with clinical stage, radiological response and overall survival in comparison with CTC enumeration. The highest detection rate of EBV cfDNA in pre-treatment samples was seen with the BamHI-W qPCR assay (89%), followed by EBNA1-dPCR (85%) and EBNA1-qPCR (67%) assays. Overall, we show that EBV cfDNA outperforms CTC enumeration in correlation with clinical outcomes of NPC patients undergoing treatment. Techniques such as dPCR and target selection of BamHI-W may improve sensitivity for EBV cfDNA detection.

Hypoxia inducible factor-1alpha and expression of vascular endothelial growth factor and its receptors in cerebral arteriovenous malformations.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its tyrosine kinase family of receptors (VEGFR) (Flt-1, Flk-1, Flt-4) have been implicated in vascular angiogenesis and remodelling in cerebral arteriovenous malformations (CAVM). In this study, we investigate the role of hypoxia inducible factor-1 (HIF-1alpha) in CAVM and its relationship to VEGF and VEGFR. METHODS: Surgical specimens from 26 patients undergoing CAVM resection were studied for HIF-1alpha , VEGF, Flt-1, Flk-1 and Flt-4. The mean age was 34.08 +/- 14.18 years. Twenty-one patients presented with intracerebral haemorrhage. RESULTS: VEGF, Flt-1 and Flt-4 were expressed in all specimens. Flk-1 was expressed in 15 of 26 patients. HIF-1alpha was expressed in 15 of 26 patients. HIF-1alpha expression was significantly associated with VEGF, Flt-1 and Flk-1 expression (p < 0.05) CONCLUSIONS: HIF-1alpha is expressed in human CAVM. The expression of HIF-1alpha is significantly related to VEGF and VEGFR expression, suggesting a possible role for its induction and role in maintaining angiogenesis and vascular remodelling.

Cerebrospinal fluid orexin in aneurysmal subarachnoid haemorrhage - a pilot study.

The hypothalamus, a vital regulator of multiple physiologic functions, is the principal source of the neuropeptide orexin, which is thought to regulate the sleep-wake cycle. As hypothalamic damage may result from aneurysmal subarachnoid haemorrhage (SAH) and be associated with a depressed conscious level, we sought to investigate whether orexin levels reflected the severity of the ictus and were of any prognostic value in SAH. CSF orexin levels from 15 patients with aneurysmal SAH were analysed for up to 14 days. The correlation between orexin and GCS, WFNS grade, Fisher grade, GOS at 6 months and hydrocephalus were ascertained. Orexin levels in 5 patients with normal pressure hydrocephalus were used as controls. Patients with GCS less than 8 on admission had undetectable orexin whilst those with a GCS of 8 or greater had measurable orexin (p < 0.05). CSF orexin levels appear to correlate with conscious level and might be a valid indicator of hypothalamic injury. As some adverse sequelae of SAH are due to hypothalamic damage, pharmacological manipulation of orexinergic neuronal pathways could lead to exciting therapeutic options in the future.

p53 point mutation is rare in meningiomas from Singaporean patients.

In Asian populations, meningiomas account for up to 35% of all central nervous system tumours, a significantly higher incidence than in Caucasian populations. While several studies have examined p53 both at the level of the gene and the protein in both benign and malignant meningiomas, its role remains controversial, particularly with regard to the discrepancy between p53 over-expression and gene mutation. We examined 19 benign meningiomas, all of which were known to over-express p53, and eight malignant meningiomas, of which three were known to over-express p53, for mutations in the p53 gene using polymerase chain reaction amplification and direct sequencing of exons 4 to 9, inclusive. Only one single mutation was detected in a benign meningioma, confirming that p53 over-expression in meningiomas is commonly found in the absence of gene mutations, and that despite the significantly higher incidence of meningiomas in some Asian populations, this is not associated with a significantly higher rate of p53 mutations.

Expression of the inhibitor of apoptosis protein survivin in benign meningiomas.

Survivin is a recently characterised inhibitor of apoptosis protein that has been implicated in the pathogenesis of several types of solid organ cancer. This study sought to describe the expression of survivin in a cohort of 90 benign meningiomas, together with the pattern of expression of other genes involved in the apoptotic process, namely bax and bcl2. Survivin expression was noted in 94% (85/90) of samples and was not correlated with the expression of either bax or bcl2 or with clinicopathological factors.

Expression of survivin in primary glioblastomas.

PURPOSE: Studies in several tumour types have suggested that the inappropriate expression of the novel inhibitor of apoptosis protein survivin may play a key role in tumourigenesis. This study presents the first immunohistochemical examination of survivin expression in glioblastomas. METHOD: The cohort consisted of 39 ethnic Chinese patients diagnosed with primary glioblastoma multiforme. Samples were archival paraffin-embedded blocks. Concomitant with examination for survivin expression, samples were also examined for over-expression of the p53 protein as well as for evidence of apoptotic cells via the terminal deoxynucleotide transferease (TdT) mediated nick end labelling (TUNEL) technique. RESULTS: Results showed that survivin was expressed in nearly 80% (31/39) of samples. Over-expression of moderate or high levels of survivin was correlated with the absence of apoptotic cells ( P=0.03). Expression of survivin and p53 was found to be significantly related ( P=0.037), and 70% (27/39) of tumours showed co-ordinate expression of p53 and survivin. CONCLUSION: Given that p53 over-expression in primary glioblastomas is predominantly detected in the absence of mutations of the gene, and that both survivin and p53 are regulated at the level of the protein by the same ubiquitin-proteosome degradation pathway, these results suggest that primary glioblastomas may occur as a result of a failure of appropriate protein degradation regulation.

Poly(adenosine diphosphate-ribose) polymerase expression in human traumatic brain injury.

OBJECT: This study was designed to elucidate the pattern of expression of poly(adenosine diphosphate-ribose) polymerase (PARP) in human pericontusional brain tissue and to correlate these findings with commonly used clinical parameters. METHODS: The expression of PARP was ascertained using immunohistochemical studies in eight specimens of human pericontusional brain tissue obtained when the patients underwent craniotomy for mass effect. The following demographic and clinical parameters were also analyzed for each patient: age, sex, postresuscitation Glasgow Coma Scale score (GCS), computerized tomography findings, intracranial pressure (ICP) recordings during the first 24 hours postsurgery, and the time interval from injury to surgery. The authors observed that PARP was present in neurons of pericontusional tissue and that it conformed to two patterns of subcellular localization; it was found either in the nucleus exclusively or in both nuclear and cytoplasmic compartments. They showed that a preponderance of cytoplasmic staining in neurons was significantly correlated with a short time interval from trauma to surgery (< or = 4 hours). There was no correlation, however, between the subcellular distribution of PARP and clinical parameters such as admission GCS score and ICP readings obtained intra- and postoperatively. CONCLUSIONS: As in earlier studies in which it has been suggested that caspase-cleaved PARP translocates to the cytoplasm during apoptosis, the authors' results indicate that apoptosis may predominate in the initial time frame after head injury. This information may well influence the timing of administration of antiapoptotic neuronal salvage agents for adjunctive therapy of head injury in the future.

Intraepidermal nerve fiber density as a marker of early diabetic neuropathy.

The purpose of the study was to reliably identify an early stage of diabetic polyneuropathy (DPN) by measuring injury to epidermal nerve fibers. We compared intraepidermal nerve fiber density (IENFD) at the ankle and thigh of 29 diabetic subjects who had no clinical or electrophysiological evidence of small- or large-fiber neuropathy to that of 84 healthy controls. The mean ankle IENFD of diabetic subjects was 9.1+/-5.0 mm and that of controls, 13.0+/-4.8 mm (P<0.001). The thigh IENFD did not differ significantly. The IENFD ratio (thigh IENFD divided by ankle IENFD) was 2.39+/-1.30 in diabetic subjects and 1.77+/-0.58 in controls (P<0.001), indicating a length-dependent reduction of IENFD in diabetics. Ankle IENFD remained significantly lower and the IENFD ratio higher in diabetic subjects after adjusting for age. Two subjects had parasympathetic dysfunction, two had retinopathy, and two early nephropathy. Age, height, weight, duration of diabetes, and average HbA1c did not influence IENFD among diabetic subjects. We used receiver operating characteristic (ROC) curves to describe and compare the utility of various threshold values of ankle IENFD and IENFD ratio for the diagnosis of early DPN. The sensitivity and specificity of diagnosing DPN using ankle IENFD of less than 10 mm were 72.4% and 76.2%, respectively. Thus, asymptomatic diabetics have a measurable, length-dependent reduction of distal epidermal nerves. Analogous to microalbuminuria in diabetic nephropathy, reliable identification and quantitation of nascent diabetic neuropathy may have potential therapeutic implications.

Determinants of epidermal nerve fiber density in normal individuals.

We studied the relationship between epidermal innervation and age, gender, height, and weight. Intraepidermal nerve fiber density (IENFD) of skin biopsies obtained from the proximal thigh and ankle of 84 normal individuals was quantified. A linear regression model was performed using IENFD at the thigh, IENFD at the ankle, and the thigh IENFD/ankle IENFD ratio, with age, gender, and height-weight interaction as predictors. An independent, negative correlation was found between age and IENFD at the ankle. No correlation was found between age and IENFD at the thigh. With increasing age the thigh IENFD/ankle IENFD ratio, a measure of the length-dependent distal-to-proximal gradient of epidermal nerve density, increased significantly. Gender, height, and body weight did not independently influence IENFD at either site. In normal individuals, distal epidermal innervation decreases in a length-dependent manner with advancing age. This must be considered when interpreting IENFD in disease states.

Expression of extracellular matrix markers in benign meningiomas.

Meningiomas have mesenchymal differentiation and studies have confirmed that meningiomas express intermediate filaments of both mesenchymal and epithelial types including vimentin and keratin. To further characterize their mesenchymal properties, particularly the role of factors requiring adhesion, extracellular matrix degradation, and migration, meningiomas were examined for a panel of extracellular matrix markers. Immunoreactivity to the matrix metalloproteinases, MMP-2 and MMP-9, and their tissue inhibitor, TIMP-1, and to an adhesion factor, galectin-3 were found in the majority of cases. The present study suggests that expression of these factors in benign meningiomas is ubiquitous and unrelated to tumor location. Therefore, these factors of the extracellular matrix may be potential targets of future therapy.

Overexpression of mdm2 and p53 and association with progesterone receptor expression in benign meningiomas.

The progesterone receptor is frequently found expressed in meningiomas at robust levels. As several studies of breast and endometrial tumors have shown an inverse correlation between progesterone receptor expression and p53 overexpression, we sought to determine if a similar relationship existed in meningiomas. As p53 may also be inactivated by the overexpression of mdm2, we examined a cohort of 90 benign meningiomas immunohistochemically for the presence of the progesterone receptor as well as overexpression of p53 and mdm2. The progesterone receptor was detected in 67% (61/90) of cases, while p53 and mdm2 overexpression were detected in 14% (13/90) and 46% (42/90) of cases, respectively. An absolute correlation was observed between the overexpression of nuclear mdm2 and overexpression of the progesterone receptor, with nuclear mdm2 overexpression being confined to progesterone receptor-positive tumors (P = 0.001). While p53 overexpression was not associated with progesterone receptor expression, a combination of mdm2 overexpression and/or p53 overexpression was significantly associated with the presence of the progesterone receptor (P = 0.025). These results suggest the existence of a novel relationship between p53 (and its regulatory control) and the presence of the progesterone receptor and, as such, may have fundamental consequences in developing progesterone receptor-targeted therapies for meningiomas.

Glioblastoma multiforme in an Asian population: evidence for a distinct genetic pathway.

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40-60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR-alpha over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for overexpression of EGFR, mdm2, p53, and PDGFR-alpha as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR-alpha were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both 'primary' and 'secondary' glioblastomas.