Daily monitoring of viral load measured as SARS-CoV-2 antigen and RNA in blood, IL-6, CRP and complement C3d predicts outcome in patients hospitalized with COVID-19.

OBJECTIVES: We hypothesized that the amount of antigen produced in the body during a COVID-19 infection might differ between patients, and that maximum concentrations would predict the degree of both inflammation and outcome for patients. METHODS: Eighty-four hospitalized and SARS-CoV-2 PCR swab-positive patients, were followed with blood sampling every day until discharge or death. A total of 444 serial EDTA plasma samples were analyzed for a range of biomarkers: SARS-CoV-2 nuclear antigen and RNA concentration, complement activation as well as several inflammatory markers, and KL-6 as a lung marker. The patients were divided into outcome groups depending on need of respiratory support and death/survival. RESULTS: Circulating SARS-CoV-2 nuclear antigen levels were above the detection limit in blood in 65 out of 84 COVID-19 PCR swab-positive patients on day one of hospitalization, as was viral RNA in plasma in 30 out of 84. In all patients, complete antigen clearance was observed within 24 days. There were definite statistically significant differences between the groups depending on their biomarkers, showing that the concentrations of virus RNA and antigen were correlated to the inflammatory biomarker levels, respiratory treatment and death. CONCLUSIONS: Viral antigen is cleared in parallel with the virus RNA levels. The levels of antigens and SARS-CoV-2 RNA in the blood correlates with the level of IL-6, inflammation, respiratory failure and death. We propose that the antigens levels together with RNA in blood can be used to predict the severity of disease, outcome, and the clearance of the virus from the body.

Detection of novel strains of cyprinid herpesvirus closely related to koi herpesvirus.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) is a devastating virus of carp. Using generic primers for the DNA polymerase and the major capsid protein genes of cyprinid herpesviruses, nucleotide sequences divergent from previously described CyHV-3 were obtained. At least 3 novel groups of putative CyHV-3-like viruses were identified, sharing 95 to 98% nucleotide identity with CyHV-3 strains. Carp carrying the CyHV-3 variants did not show clinical signs consistent with CyHV-3 infection and originated from locations with no actual CyHV-3 outbreaks. These strains might represent low- or non-pathogenic variants of CyHV-3.

Phase II trial of delta-tocotrienol in neoadjuvant breast cancer with evaluation of treatment response using ctDNA.

Neoadjuvant treatment of breast cancer is applied to an increasing extent, but treatment response varies and side effects pose a challenge. The vitamin E isoform delta-tocotrienol might enhance the efficacy of chemotherapy and reduce the risk of side effects. The aim of this study was to investigate the clinical effect of delta-tocotrienol combined with standard neoadjuvant treatment and the possible association between detectable circulating tumor DNA (ctDNA) during and after neoadjuvant treatment with pathological treatment response. This open-label, randomized phase II trial included 80 women with newly diagnosed, histologically verified breast cancer randomized to standard neoadjuvant treatment alone or in combination with delta-tocotrienol. There was no difference in the response rate or frequency of serious adverse events between the two arms. We developed a multiplex digital droplet polymerase chain reaction (ddPCR) assay for the detection of ctDNA in breast cancer patients that targets a combination of two methylations specific for breast tissue (LMX1B and ZNF296) and one cancer specific methylation (HOXA9). The sensitivity of the assay increased when the cancer specific marker was combined with the ones specific to breast tissue (p < 0.001). The results did not show any association between ctDNA status and pathological treatment response, neither at midterm nor before surgery.

Nizp1 zinc finger protein localization is determined by SCAN-domain inclusion regulated through alternative splicing.

The NSD1 histone methyl transferase is involved in childhood acute myeloid leukemia and the outgrowth disorders Sotos and Weaver syndromes. NSD1 is a transcriptional co-repressor for the zinc finger protein Nizp1 (also abbreviated Zfp496 and Zkscan17). Nizp1 includes a SCAN-domain, a KRAB-domain, four C2H2 Krüppel related zinc fingers, and a C2HR transcriptional repression and protein interaction domain required for NSD1 interaction. In this study we have identified alternative splicing of the Nizp1 gene resulting in transcripts encoding Nizp1 protein isoforms with a short N-terminal deletion or a SCAN-domain deletion. The alternative Nizp1 transcripts are expressed in lower levels relative to the canonical Nizp1 transcript. The Nizp1 SCAN-domain mediates Nizp1 self-association but lacks intrinsic transcriptional activating or repressing capacity and has no influence on the transcriptional repression activity of Nizp1 in reporter assays. Sub-cellular localization analysis showed that a fraction of Nizp1 localizes to CBP nuclear bodies and that the SCAN-domain is required for the localization to nuclear bodies. The presented results show that alternative splicing is a functional mechanism to generate Nizp1 protein isoforms with different SCAN-domain compositions and accordingly different sub-cellular localizations.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.

The NSD3L histone methyltransferase regulates cell cycle and cell invasion in breast cancer cells.

NSD3/WHSC1L1 histone methyltransferase gene aberrations are observed in leukemia and in breast and lung carcinomas, suggesting that NSD3 is implicated in carcinogenesis. In this study we examined in human breast cancer cells the NSD3L isoform which contains the catalytic histone methyltransferase SET-domain. siRNA directed depletion of NSD3L followed by genome-wide microarray analysis identified NSD3L regulated genes which could be functionally linked to cellular signaling pathways such as cell growth, cell cycle, cell motility, transcription, and apoptosis. Notably up-regulated genes are the cell cycle regulators E2F2 and Arl2. In accordance with a function of NSD3L in cell cycle regulation NSD3L depletion resulted in an increase in the number of cells in the S and G2/M cell cycle phases. Moreover, NSD3L depletion increased the invasiveness of MDA-MB-231 breast cancer cells indicating that NSD3L normally restrain cellular metastatic potential. Together the presented data indicates that NSD3L is a candidate tumor suppressor.

Identification of a novel vimentin promoter and mRNA isoform.

The intermediate filament protein vimentin is involved in a variety of cellular functions both during the normal developmental processes and in human malignancies. We here describe the identification of an alternative vimentin transcript initiating upstream for the canonical vimentin gene promoter and spliced using the vimentin promoter sequence as an intron. Expression analysis showed that the alternative vimentin promoter had the same expression profile as the canonical vimentin gene promoter. The presented data suggest that alternative promoter usage and alternative splicing could be regulatory mechanisms participating in vimentin gene regulation.

Cellular parkin mutants are soluble under non-stress conditions.

The parkin gene encodes an E3 ubiquitin ligase and loss of function mutations herein are the most frequent cause of early-onset Parkinson's disease. Reports have suggested that aggregation of mutant protein is the cause of the loss of function. We established stably transfected SH-SY5Y dopaminergic cell lines expressing wild-type and mutant parkin proteins. All the mutant proteins were soluble but could be rendered insoluble by subjecting the cellsto stress by proteasomal inhibition, treatment with oxidants and upon transient expression of the mutant proteins. A functional assay demonstrated that the R42P mutant retained functional activity in contrast to the W453stop mutant. Accordingly, the functional impairment by the mutations is not simply caused by turning the proteins insoluble.

A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo.

Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.

Caspase-mediated parkin cleavage in apoptotic cell death.

The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.

Caspase-1 and caspase-8 cleave and inactivate cellular parkin.

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.

Parkin is recruited into aggresomes in a stress-specific manner: over-expression of parkin reduces aggresome formation but can be dissociated from parkin's effect on neuronal survival.

Parkinson's disease (PD) is characterized by loss of dopamine neurons in the substantia nigra and the presence of cytoplasmic inclusions known as Lewy bodies (LBs). Mutations in parkin cause autosomal recessive juvenile parkinsonism (AR-JP) that is distinct from sporadic PD by the general absence of LBs. Several studies have reported that parkin is present in LBs of sporadic PD but the role of parkin in LB formation is unclear. Aggresomes are perinuclear aggregates representing intracellular deposition of misfolded protein. LBs and aggresomes have been reported to share a common biogenesis. We have investigated the role of parkin in aggresome formation. In human SH-SY5Y neuroblastoma cells we observe that endogenous parkin is present in aggresomes induced by a variety of stresses including dopamine, proteosome inhibition and a pro-apoptopic stimulus. We show that vimentin is invariably collapsed around the aggresome but that the detection of ubiquitin is variable depending on the stress. We show that cells that stably over-express human wild-type parkin form fewer aggresomes upon stress compared to cells that express vector alone whereas over-expression of AR-JP causing mutants of parkin have no effect on stress-induced aggresome formation. Finally, we show that the prevention of aggresome formation by over-expression of wild-type parkin is not always associated with a beneficial effect on neuronal survival. Our findings suggest that parkin is important for aggresome formation in human neuronal cells and may lead to a better understanding of the biogenesis of LBs in sporadic PD.