Tumor expression of human chorionic gonadotropin beta mRNA and prognosis of prostate cancer treated by radical prostatectomy

The beta subunit of human chorionic gonadotropin (hCG beta) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p <.0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p <.0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.Peer reviewe

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N=571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted invitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.Peer reviewe

No Tissue Expression of KRAS or BRAF Mutations in 61 Adult Patients Treated for Esophageal Atresia in Early Childhood

Background Previous studies have reported an association among esophageal atresia (EA), Barrett's esophagus, and esophageal adenocarcinoma later in life. Objective The objective of the article is to evaluate KRAS and BRAF mutations as potential genetic markers for early detection of malignant transformation, we used an ultrasensitive technique to detect tissue expression of KRAS and BRAF mutations in endoscopic biopsies from 61 adult patients under follow-up after treatment for EA. Materials and Methods RNA was extracted from 112 fresh-frozen endoscopic tissue biopsies from 61 adult patients treated for EA in early childhood. RNA was reverse transcribed using the extendable blocking probe reverse transcription method. KRAS codons 12 and 13, as well as BRAF mutations were detected by quantitative polymerase chain reaction. Results No mutations of KRAS codon 12, KRAS codon 13, or BRAF were found in 112 endoscopic biopsy samples from 61 patients. Conclusion Despite the presence of histological findings indicating long-standing gastroesophageal reflux in 25%, as well as symptomatic gastroesophageal reflux in more than 40%, there was no detectable tissue expression of KRAS or BRAF mutations in this cohort of patients.Peer reviewe

An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants

Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5-1000 mu g/L. The limit of quantitation (LOQ) was 0.5 mu g/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98-1.01, n = 11), 0.55 (95% CI 0.43-0.61, n = 14), and 0.05 (range 0.04-0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.Peer reviewe

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

Peer reviewe

Immuno-modulating properties of saliphenylhalamide, SNS-032, obatoclax, and gemcitabine

Influenza A viruses (IAVs) impact the public health and global economy by causing yearly epidemics and occasional pandemics. Several anti-IAV drugs are available and many are in development. However, the question remains which of these antiviral agents may allow activation of immune responses and protect patients against co- and re-infections. To answer to this question, we analysed immuno-modulating properties of the antivirals saliphenylhalamide (SaliPhe), SNS-032, obatoclax, and gemcitabine, and found that only gemcitabine did not impair immune responses in infected cells. It also allowed activation of innate immune responses in lipopolysaccharide (LPS)- and interferon alpha (IFN alpha)-stimulated macrophages. Moreover, immuno-mediators produced by gemcitabine-treated IAV-infected macrophages were able to prime immune responses in non-infected cells. Thus, we identified an antiviral agent which might be beneficial for treatment of patients with severe viral infections. (C) 2015 The Authors. Published by Elsevier B.V.Peer reviewe

A Phase II Trial of a Personalized, Dose-Intense Administration Schedule of (177)Lutetium-DOTATATE in Children With Primary Refractory or Relapsed High-Risk Neuroblastoma-LuDO-N

Background:& nbsp;Half the children with high-risk neuroblastoma die with widespread metastases. Molecular radiotherapy is an attractive systemic treatment for this relatively radiosensitive tumor. I-131-mIBG is the most widely used form in current use, but is not universally effective. Clinical trials of (177)Lutetium DOTATATE have so far had disappointing results, possibly because the administered activity was too low, and the courses were spread over too long a period of time, for a rapidly proliferating tumor. We have devised an alternative administration schedule to overcome these limitations. This involves two high-activity administrations of single agent Lu-177-DOTATATE given 2 weeks apart, prescribed as a personalized whole body radiation absorbed dose, rather than a fixed administered activity. "A phase II trial of (177)Lutetium-DOTATATE in children with primary refractory or relapsed high-risk neuroblastoma - LuDO-N " (EudraCT No: 2020-004445-36, Identifier: NCT04903899) evaluates this new dosing schedule.& nbsp;Methods:& nbsp;The LuDO-N trial is a phase II, open label, multi-center, single arm, two stage design clinical trial. Children aged 18 months to 18 years are eligible. The trial is conducted by the Nordic Society for Pediatric Hematology and Oncology (NOPHO) and it has been endorsed by SIOPEN (). The Karolinska University Hospital, is the sponsor of the LuDO-N trial, which is conducted in collaboration with Advanced Accelerator Applications, a Novartis company. All Scandinavian countries, Lithuania and the Netherlands participate in the trial and the UK has voiced an interest in joining in 2022.& nbsp;Results:& nbsp;The pediatric use of the Investigational Medicinal Product (IMP) Lu-177-DOTATATE, as well as non-IMPs SomaKit TOC (R) (Ga-68-DOTATOC) and LysaKare (R) amino acid solution for renal protection, have been approved for pediatric use, within the LuDO-N Trial by the European Medicines Agency (EMA). The trial is currently recruiting. Recruitment is estimated to be finalized within 3-5 years.& nbsp;Discussion:& nbsp;In this paper we present the protocol of the LuDO-N Trial. The rationale and design of the trial are discussed in relation to other ongoing, or planned trials with similar objectives. Further, we discuss the rapid development of targeted radiopharmaceutical therapy and the future perspectives for developing novel therapies for high-risk neuroblastoma and other pediatric solid tumors.Peer reviewe

Framtidens studentboende : En studie om studentens behov och krav på sitt framtida boende med ett gestaltningsförslag i passivhusstandard

In Stockholm, and other metropolitan areas, the lack of housing and especially student housing have been long-standing. Boverket says this is due to an strong population growth in combination with a relative low house building. The demand of higher education will keep raising, despite the fact that in a few years will we have passed the peek of youths. This while Stockholm municipality wants to set the goal; in year 2015 all newly built premises and housings should have an energy consumption per square meter that corresponds to the energy consumption of an passive house according to Åfreds (2011).The purpose of this thesis is to propose changes and improvements for the housing for students, socially, academically and environmentally to and examine how it could be performed with passive house technology for Stiftelsen Stockholms Studentbostäder. The thesis identifies wich kind of housing and wich functions students demands regarding future student housing and how much they are willing to pay for this variables. The goal of the thesis is through research and previous studies present a design proposal for a conceptually, energy efficient and flexible student housing at a selected site, Solna, in Stockholm. The design proposal should contain drawings and descriptions of the accommodation.To identify what the students demands when it comes to new student housings, two surveys have been designed and distributed to students in Stockholm. The surveys were quantitatively designed and was presented in bar charts, but analyzed and interpreted from a quantitative approach. A random sample of 60 respectively 100 persons at KTH in Stockholm were questioned. This have supplemented with SCB’s studie from 2004 to get a broader and a more in depth perception.A theoretical background was conceived with basic theory which highly affects the research and the last part in this thesis is to produce a design proposal for the future of student housings.The interpretation and reporting of the result was performed by drawing up tables in Excel and then make an interpretation of the results that are in the report's analysis section. The results were analyzed and a "basic accommodation" was developed from this.According to the author’s investigation is the typical student who have respondent between 19-25, single and living either in a student residence or in an ordinary apartment/house. The students want the student housing should be close to good public transport (92%), have a reasonable rent (97.5%) and are happy to be close to university (68%).In the building want the students to have access to a social room and study room. But these rooms must not take up too much space and affect the rent to any great extent. The rooms are still important in the home. From the social perspective wanted the student to be largely social in their living and they study mostly in groups. 67% of the students pursue their self-study at home, according to SCB's survey from 2004.The students ranked four accommodations i the survey. As the first choice they ranked the student living in "a room with a kitchenette" (45%), followed by "a room and kitchen" (29%), "corridor room" (15%) and "double room apartment" (13%).The student would be able to cook their meals in their apartment, in a fully equipped kitchen. 60% consider themselves to be in need of a desk in his apartment, but many choose to study on the sofa or dining table. They also pay for a normal sized bathroom, handicapped accessible, than compromising on the area for the function. 120 cm wide bed was wanted as much as 95% of respondents, and even sleep traditionally. Inventory and storage was also very important. Whether the apartment is furnished or not, 80% said it was unimportant / very unimportant. The student is also environmentally friendly and would consider lowering the rent by living eco-friendly.In 2004, 91% of the students in Stockholm said the maximum rent per month was 3500 Swedish crowns. That's about 4100 Swedish crowns / month for the rent with the current monetary measure and the student funds from the government. The majority were willing to pay up to 5200 Swedish crowns/month for an apartment at 24 sqm. 55% of students are willing to share their room in an apartment of 24 sqm.The energy calculation on the actual student housing, according to a rough estimate in PHPP 2007, shows a specific heat at:• Stockholm: 8 kWh / (M2A)• Kiruna: 20 kWh / (M2A)• Lund: 7 kWh / (M2A)The main conclusion is that the future of student housing in Stockholm will be located at the “right place”, the students would rather pay more for a quality accommodation than they want to compromise on the features of student housing and that it should be performed in the passive house standard.Validerat; 20120511 (anonymous

Multiple Heat Pulses during PCR Extension Enabling Amplification of GC-Rich Sequences and Reducing Amplification Bias

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele