Subacute Sclerosing Panencephalitis

Total yeast RNA was purified as described [109] (link). Primer extension analysis was performed exactly as described (http://labs.fhcrc.org/hahn/Methods/mol_bio_meth/primer_ext.html) [110] (link) with the following modifications. Total RNA used was 30 µg, and in the case of more dilute RNA samples, volumes of reactions were increased by 50% to accommodate greater volume of sample. Reverse transcriptase (RT) was M-MLV RT from either Life Technologies or Fermentas. RT synthesis reactions were supplemented with 1 µl RNAse Inhibitor (Fermentas). Extension products were separated on either 7% or 10% acrylamide-bisacrylamide gels (19∶1 ratio) containing 1× TBE and 7 M Urea. Northern blotting was performed essentially as described in manual for GeneScreen hybridization membranes (Perkin-Elmer) with the following modifications. RNA samples (20 µg) were prepared in NorthernMax loading buffer (Ambion/AB). Prehybridization solution did not contain SSPE or SSC buffers, but contained 5× Denhardt's solution, 50 mM Tris-HCl pH 7.5, 1 M NaCl, 0.1% sodium pyrophosphate, 0.1% SDS instead of 1%, 10% Dextran Sulfate, 50% formamide, and 500 µg/ml sheared/denatured salmon sperm DNA. Probes for northern blots were radiolabeled using α-³²P-dATP by random priming using the Decaprime II kit (Ambion) according to manufacturer's instructions. Blots were washed at twice for 10 minutes each wash at 42°C with 2× SSC, 0.5% SDS, then twice at 67°C with 5× SSC, 0.5% SDS for 30 minutes each wash, then twice in 0.2× SSC for 30 minutes each wash at room temperature. Primer extension gels and northern blots were visualized by phosphorimaging (GE Healthcare or Bio-Rad) and quantified using ImageQuant 5.0 (GE) or Quantity One (Bio-Rad) software, with data exported to Microsoft Excel for management. Oligo sequences for site-directed mutagenesis, primer extension analysis, amplification of DNA for Northern blotting and in vitro transcription are available upon request.

Genomic DNA was extracted from freshly cultured bacteria. Cells were resuspended in 500 µL TE buffer(pH 8. 0), then heated in 95°C water bath for 1 hour. After centrifugation of cellular debris, DNA in the supernatent served as PCR template. Spoligotyping was used to identify the genotype of TB strain in the DR locus as described previously [10] (link). A commercially available kit (Isogen Bioscience BV, Maarssen, The Netherlands) was used as described by the manufacturer. All chromosomal DNA was amplified with primers DRa (5′-CCGAGAGGGGACGGAAAC-3′) and DRb (5′-GGTTTTGGGTCTGACGAC-3′). Then the amplified products were hybridized with the membrane. After washing with 2×SSPE solution (360 mM NaCl, 20 mM NaH₂PO₄, 2 mM EDTA [pH 7.2]) supplemented with 0.5% SDS, the membrane was hybridized with streptavidin-peroxidase conjugate. The final image was detected with a chemiluminescence system, including the ECL detection liquid (Amersham, Buckinghamshire, United Kingdom) and ECL-Hyperfilm (Kodak, Rochester, NY). Beijing genotype strains were defined with the pattern that hybridized to all of the last nine spacer oligonucleotides (spacers 35 to 43), and Beijing-like genotype strains were ones that hybridized to only some of the last nine spacers.

Denaturing formaldehyde agarose gel electrophoresis was performed as described in reference 3 (link). In brief, 2% agarose gels containing 6% deionized formaldehyde were prepared in 1× MOPS running buffer, pH 7 (40 mM MOPS, 10 mM NaOAc, 1 mM EDTA, pH 8.0). The RNA sample (5 μL) was mixed with 15 μL RNA loading buffer (8 M urea, 6% formaldehyde, 1× MOPS running buffer, 0.05% bromophenol blue) and loaded onto the gel. Electrophoresis was performed in MOPS running buffer with circulation at 100 V for approximately 10 min, followed by 125 V until the dye front had migrated approximately 9 cm. Capillary transfer of separated RNA onto Hybond N+ membranes was performed overnight in 2× SSC (300 mM NaCl; 30 mM trisodium citrate dihydrate; pH 7.0) using a 1% agarose gel in 2× SSC as the buffer reservoir. After tRNA was cross-linked to the membrane by irradiation with 0.12 J/cm² of UV light in a UVC 500 UV crosslinker (Hoefer). Membranes were prehybridized in hybridization buffer (90 mM NaCl, 50 mM NaH₂PO₄, pH 7.7, 5 mM EDTA, 5× Denhardt’s solution, 0.5% [wt/vol] SDS, 100 mg/mL sheared, denatured salmon sperm DNA) at 42°C for at least 1 h before addition of radioactively labeled DNA oligonucleotide and hybridization at 42°C overnight. After hybridization, membranes were washed at least three times in wash buffer (2× SSC, 0.1% SDS) at RT for 1 to 2 h, and radiation was detected by phosphor imaging in an Amersham Typhoon. For reprobing, hybridized oligonucleotides were removed by washing in boiling stripping buffer (0.1× SSPE, 0.5% SDS) until no remaining radiation was detected. Oligonucleotide probes were 5′ end labeled with [³²P] by incubation of 150 pmol of oligonucleotide (1.5 μL of a 100 μM stock) with 10 U T4 polynucleotide kinase (PNK; Thermo Scientific) and 60 μCi ɣ-[³²P]-ATP (6,000 Ci/mmol; Perkin Elmer) in a total volume of 10 μL at 37°C for 1 h. After 1 h, additional 10 U of PNK was added and incubation continued for 1 h.
Normalization of tRNA signals was performed as described in reference 11 (link). For each lane, the bands corresponding to the target tRNA and the spike-in tRNA^selC were quantified using ImageQuant TL 8.2 image analysis software (GE Healthcare). The obtained target tRNA values were corrected by subtraction of the signal originating from endogenous expression of the same tRNA in the spike-in cell sample. The corrected values were then normalized by dividing the corrected value by the value of tRNA^selC from the same lane and plotted relative to the average of three samples taken during balanced growth.
tRNA levels in radiolabeled RNA samples obtained by hot phenol extraction were determined in the same manner, with the following exceptions. Prior to hybridization with oligonucleotide probes, the [¹⁴C] signal was visualized by phosphor imaging. During exposure, blots hybridized with [³²P]-labeled oligonucleotide probes were covered with several layers of plastic sheets to minimize exposure of the storage phosphor screen to radioactivity originating from [¹⁴C]-labeled RNA. For normalization, the signal intensity of the target tRNA band was divided by the [¹⁴C]-signal of the entire tRNA fraction of the same lane, which was shown to be stable by the radiolabeling approach described above. The statistical significance of differences between individual time points after starvation/treatment and the steady-state level was assessed where applicable (n ≥ 3) by a two-tailed Student’s t test assuming unequal variances. To do so, the mean and standard deviation of all available steady-state samples (routinely three steady-state samples per sample series) were used.

Southern blot analysis of DM1 myoblasts was performed by the CHEO genetics clinic. Genomic DNA was extracted using DNA mini kit (Qiagen, Toronto, ON, Canada), digested with CutSmart EcoRI-HF (New England BioLabs, Ipswich, MA, USA) for 5 h to overnight, and resolved on 0.6% agarose gel alongside DNA from an individual known to be void of the DM1 genotype (negative control) and DNA from individuals known to have DM1 with pre-established repeat sizes (positive controls); these negative and positive control samples served as representative controls used for diagnostic purposes at the CHEO genetics clinic. The gel was stained with 0.6 µg/mL ethidium bromide in 0.5× TBE for 30 min at room temperature (RT) and imaged. Subsequently, the gel was denatured in a solution of 0.4 M NaOH and 1.5 M NaCl for 210 min under agitation and transferred to Biodyne B nylon membrane (Thermo Scientific, Waltham, MA, USA) in 20× saline sodium citrate (SSC) buffer overnight at RT. Post-transfer, the membrane was neutralized in 0.2 M Tris pH 7.5 and 2× SSC buffer for 15 min at RT, baked between Whatman 3 M paper for 2 h at 80 °C, and pre-hybridized in a PEG hybridization solution of 10% PEG Glycol 8000, 1.5× SSPE, and 7% SDS for 1 h at 65 °C under rotation. Hybridization probes were radioactively labeled with 32P using Prime-It II Random Primer Labeling Kit (Agilent, Santa Clara, CA, USA), purified using Sephadex G-50 Nick column (GE Lifesciences, Chicago, IL, USA), and verified to have radioactivity of 0.3 × 10 mR/h or more per membrane. The radioactive probe was then mixed with 2 mg/mL salmon sperm DNA. It was denatured at 100 °C for 5 min, diluted approximately 1/10 in PEG hybridization buffer, and used to hybridize the membrane at 65 °C for 3 h to overnight under rotation. Following hybridization, the membrane was washed in a solution of 0.1× SSC and 0.1% SDS for 2 × 15 min at RT under agitation. Hybridized membranes were imaged using X-ray film.

The categorical variables were reported as frequency counts (percentages) and compared using the chi-square test (two-sided) and Fisher’s Exact Test (two-sided). The continuous variables were reported as mean and standard deviation (or median with interquartile range, if not normally distributed), and compared using the Student’s t-test. The incidence rates of all the VTE-related outcomes (PE recurrences, DVT, major bleeding, or death) were calculated as a number of events per 100 patient years. The incidence rate of symptomatic PE recurrences and all-cause death were compared between patients presenting with asymptomatic SSPE versus symptomatic SSPE using Cox proportional hazard regression models. The hazard ratios (HR) with the corresponding 95% confidence intervals (CI) were used as the effect measure. We used competing-risk analysis for death to compare the risk of developing symptomatic PE recurrences between subgroups. The covariates included in the adjusted models were those considered clinically significant or those for which a significant difference (a threshold p-value of <0.1) was found. The variables entered in the models included sex, age, body weight, risk factors for SSPE (i.e., active cancer, transient risk factors, or unprovoked), diabetes, hypertension, chronic heart failure, chronic lung disease, prior myocardial infarction, ischemic stroke or peripheral artery disease, recent (<30 days before) major bleeding, and discontinuation of anticoagulant therapy.
All the analyses were conducted using the statistical IBM SPSS software v25.0 (SPSS Inc., Chicago, IL, USA).