Linda Jansson

Delta-Like 1 (Dlk-1), a Novel Marker of Prostate Basal and Candidate Epithelial Stem Cells, Is Downregulated by Notch Signalling in Intermediate/Transit Amplifying Cells of the Human Prostate

BACKGROUND There is a lack of understanding of the processes that regulate differentiation in the prostate. OBJECTIVE To determine localisation, activity, and regulation of cytodifferentiation-modulatory proteins in the human adult prostate. DESIGN, SETTINGS, AND PARTICIPANTS Eighteen volunteering patients with organ-confined prostate cancer were prospectively enrolled at a single university hospital. INTERVENTION All patients underwent radical prostatectomy, and normal/benign tissue was excised and obtained from the transition zone. MEASUREMENTS Expression and activity of Notch-protein family members, including the Notch-homologous protein Delta-like 1 (Dlk-1/Pref1), were investigated immunohistochemically in normal/benign tissue and explant cultures. The effect of the Notch inhibitor L-685,458 on Dlk-1 expression and cell number was investigated in primary cell cultures, and data were analysed with Student t test. RESULTS AND LIMITATIONS Mature luminal cells were found to co-express Notch-1 and its ligand Jagged1, but epithelia in normal/benign tissue showed no active Notch signalling. The basal cell layer, rare candidate epithelial stem cells, and a subpopulation of neuroendocrine cells expressed the differentiation protein Dlk-1. In explant cultures, luminal cells and Jagged1 expression were lost, whereas intermediate cells downregulated Dlk-1 concomitant with Notch-1 upregulation and activation. Notch inhibition in primary cell cultures led to lower cell densities (p<0.001) and suppressed downregulation of Dlk-1. This is a small study; current results need to be confirmed in larger investigations. CONCLUSIONS We demonstrate that Notch-1 is upregulated in differentiation of prostate epithelia, and that the novel prostate progenitor marker Dlk-1 is downregulated by Notch signalling in intermediate cells. The identification of Dlk-1-expressing candidate stem and neuroendocrine cells suggests a hierarchical relationship.

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction

Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.

Zebularine induces long-term survival of pancreatic islet allotransplants in streptozotocin treated diabetic rats.

Background Coping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed. Methodology/Principal Findings The DNA demethylating agent Zebularine, previously demonstrated to induce expression of the genes for the immunosuppressive enzymes indolamine-2,3-deoxygenase-1 (IDO1) and kynureninase of the kynurenine pathway, is tested for capacity to suppress rejection of allotransplants. Allogeneic pancreatic islets from Lewis rats were transplanted under the kidney capsule of Fischer rats previously made diabetic by a streptozotocin injection (40 mg/kg). One group was treated with Zebularine (225 mg/kg) daily for 14 days from day 6 or 8 after transplantation, and a control group received no further treatment. Survival of the transplants was monitored by blood sugar measurements. Rats, normoglycemic for 90 days after allografting, were subjected to transplant removal by nephrectomy to confirm whether normoglycemia was indeed due to a surviving insulin producing transplant, or alternatively was a result of recovery of pancreatic insulin production in some toxin-treated rats. Of 9 Zebularine treated rats, 4 were still normoglycemic after 90 days and became hyperglycemic after nephrectomy. The mean length of normoglycemia in the Zebularine group was 67±8 days as compared to 14±3 days in 9 controls. Seven rats (2 controls and 5 Zebularine treated) were normoglycemic at 90 days due to pancreatic recovery as demonstrated by failure of nephrectomy to induce hyperglycemia. Conclusions/Significance Zebularine treatment in vivo induces a long-lasting suppression of the immune destruction of allogeneic pancreatic islets resulting in protection of allograft function for more than 10 weeks after end of treatment.

Improved Detection of Norovirus and Hepatitis A Virus in Surface Water by Applying Pre-PCR Processing

Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with Cq shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.

Evaluation and modification of lanthanum-based flocculation for isolation of bacteria from water samples

Highlights • A published lanthanum-based flocculation protocol is evaluated for four bacterial species• The success of lanthanum-based flocculation is determined by both the bacterial species and the nature of the water sample• Addition of 20 mM bicarbonate significantly improve the flocculation efficiency for tap water

Blending DNA binding dyes to improve detection in real-time PCR

Highlights • Humic acid quenches the fluorescence of the dsDNA binding dyes EvaGreen, ResoLight, SYBR Green I and SYTO9.• A four-dye blend containing the dyes above provides maximum fluorescence signals in presence and absence of humic acid.• Blending complementary dyes enables improved qualitative detection of DNA in samples with high amounts of humic acid.• Humic acid interactions with dsDNA binding dyes lower the Tm of amplicons, thereby disturbing melt curve analysis.