51 research outputs found
Novel methods to study genomic fragility and structural variation
DNA double-strand breaks (DSBs) are major DNA lesions that when repaired
unfaithfully can give rise to loss of genetic information, chromosomal rearrangements
such as insertions/deletions (indels) and copy number alterations (CNAs),
which in turn lead to genomic instability that is characteristic of almost all cancer
types. In this context, it is thought that genomic instability has critical roles in
cancer initiation, progression and intra-tumor heterogeneity (ITH). DSBs have
also been exploited for genome-editing purposes where using different CRISPR
(clustered regularly interspaced short palindromic repeats) systems, one can create
DSBs in the target DNA to alter sequences and modify gene function. However, this
approach is not without drawbacks, as DSBs can be created at sites other than the
intended target (known as off-target effects), which can potentially be mutagenic.
Therefore, given the importance of DSBs in genomic instability, their role in generation
of CNAs and genome-editing technologies, it is of great interest to determine
genomic locations of DSBs and their frequency along the genome, together with
DNA copy number profiling. Thus, the focus of this thesis was to develop molecular
tools for detection and quantification of DSBs with single-nucleotide resolution
in different model systems, in combination with the development of technologies
for DNA copy number profiling, by which we can collectively understand the
biology behind DSBs, their links to CNAs in the context of cancer and assess the
safety profile of CRISPR systems for therapeutic applications.
In Paper I, we developed BLISS (Breaks Labeling In Situ and Sequencing) as a
quantitative method enabling genome-wide DSB profiling. We showed that BLISS
accurately identified both endogenous and drug-induced DSBs genome-wide, even
in samples of a few thousand cells and in single tissue sections. Additionally, we
demonstrated that BLISS is a powerful tool to measure the off-target activities
of Cas9 and Cpf1 CRISPR systems, and indeed we found that Cpf1 was more
specific than Cas9.
In Paper II, using BLISS-generated DSB data from cell lines, we modeled the
contribution of genetic and epigenetic features in shaping the cancer fragility, and
made predictions of the frequency of expected breaks across the human genome.
We constructed random forest regression models from four DSB datasets and found
that the most influential feature in DSB frequency prediction is replication timing
across all models. In addition, we noticed that open chromatin at transcriptionally
active genes and associated regulatory factors have the largest influence on the
frequency of DSBs than transcription per se.
In Paper III, we developed CUTseq, which builds on the design of BLISS from
Paper I, and can be used for gDNA barcoding and amplification to generate
multiplexed DNA sequencing libraries for performing reduced representation
sequencing of DNA samples extracted from cell lines, FFPE tissue sections or
small sub-regions thereof. We demonstrated the applicability of CUTseq for CNA
profiling, and showed that CUTseq can reproducibly detect a considerable fraction
of high-confidence single nucleotide variants (SNVs) that were also detected by
a standard exome capture method. Finally, we demonstrated that CUTseq can
be applied for multi-region tumor sequencing to assess ITH of CNA profiles of
multiple-small regions of a single FFPE tissue sections of primary and metastatic
breast cancer lesions
A review of methods to estimate the visibility factor for bias correction in network scale-up studies
Network scale-up is an indirect size estimation method, in which participants are questioned on sensitive behaviors of their social network members. Therefore, the visibility of the behavior affects the replies and estimates. Many attempts to estimate visibility have been made. The aims of this study were to review the main methods used to address visibility and to provide a summary of reported visibility factors (VFs) across populations. We systematically searched relevant databases and Google. In total, 15 studies and reports that calculated VFs were found. VF calculation studies have been applied in 9 countries, mostly in East Asia and Eastern Europe. The methods applied were expert opinion, comparison of NSU with another method, the game of contacts, social respect, and the coming-out rate. The VF has been calculated for heavy drug users, people who inject drugs (PWID), female sex workers (FSWs) and their clients, male who have sex with male (MSM), alcohol and methamphetamine users, and those who have experienced extra-/pre-marital sex and abortion. The VF varied from 1.4% in Japan to 52.0% in China for MSM; from 34.0% in Ukraine to 111.0% in China for FSWs; and from 12.0% among Iranian students to 57.0% in Ukraine for PWID. Our review revealed that VF estimates were heterogeneous, and were not available for most settings, in particular the Middle East and North Africa region, except Iran. More concrete methodologies to estimate the VF are required
Care Seeking Patterns of STIs-Associated Symptoms in Iran: Findings of a Population-Based Survey
Background: Understanding the prevalence of symptoms associated with sexually transmitted infections (STIs)
and how care is sought for those symptoms are important components of STIs control and prevention. People’s
preference between public and private service providers is another important part of developing a well-functioning
STIs surveillance system.
Methods: This cross-sectional survey was carried out in spring 2011, using a nonrandom quota sample of 1190
participants (52% female) in 4 densely-populated cities of Tehran, Kerman, Shiraz, and Babol. Two predictive
logistic regression models were constructed to assess the association between the socio-demographic determinants
(independent variables) and the dependent variables of history of STIs-associated symptom and seeking care.
Results: Around 57% (677 out of 1190; men: 29.70% and women: 81.80%) had experienced at least one STIsassociated
symptom during the previous year. History of experiencing STIs-associated symptoms among men, was
negatively significantly associated with older age (adjusted odds ratio [AOR]=0.34, CI 95%: 0.17-0.67). Women
who were married, in older ages, and had higher educations were more likely to report a recent (past year) STIs
symptom, however all were statistically insignificant in both bivariate and multivariable models. Among those
who have had STIs-associated symptoms in the last year, 31.15% did nothing to improve their symptoms, 8.03%
attempted self-treatment by over-the-counter (OTC) medications or traditional remedies, and 60.93% sought
care in health facilities. In both bivariate and multivariable analyses, care seeking among men was insignificantly
associated with any of the collected demographic variables. Care seeking among women was positively significantly
associated with being married (AOR=2.48, 95% CI: 1.60-3.84).
Conclusion: The reported prevalence of STIs-associated symptoms among our participants is concerning. A
considerable number of participants had delayed seeking care and treatment or self-medicated. People should be
informed about their sexual health and the consequences of delaying or avoiding seeking care for STIs. Participants
preferred seeking care at private sectors which calls for engaging both public and private health sectors for reporting
and following up STIs cases
BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049
Spatially resolved clonal copy number alterations in benign and malignant tissue
Publisher Copyright: © 2022, The Author(s).Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.Peer reviewe
Spatially resolved clonal copy number alterations in benign and malignant tissue
Publisher Copyright: © 2022, The Author(s).Defining the transition from benign to malignant tissue is fundamental to improving early diagnosis of cancer1. Here we use a systematic approach to study spatial genome integrity in situ and describe previously unidentified clonal relationships. We used spatially resolved transcriptomics2 to infer spatial copy number variations in >120,000 regions across multiple organs, in benign and malignant tissues. We demonstrate that genome-wide copy number variation reveals distinct clonal patterns within tumours and in nearby benign tissue using an organ-wide approach focused on the prostate. Our results suggest a model for how genomic instability arises in histologically benign tissue that may represent early events in cancer evolution. We highlight the power of capturing the molecular and spatial continuums in a tissue context and challenge the rationale for treatment paradigms, including focal therapy.Peer reviewe
Modeling double strand break susceptibility to interrogate structural variation in cancer
Abstract Background Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs). Results We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers. Conclusions We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors
Lead time and ordering cost reductions in budget and storage space restricted probabilistic inventory models with imperfect items
Regarding today’s business environment restrictions, one of significant concern of
inventory manager is to determine optimal policies of inventory/production systems under
some restrictions such as budget and storage space. Therefore here, a budget constraint on
total inventory investment and a maximum permissible storage space constraint are added
simultaneously to a stochastic continuous review mixed backorder and lost sales inventory
system. This study also assumes that the received lot may contain some defective units
with a beta-binomial random variable. Two lead time demand (LTD) distribution approach are
proposed in this paper, one with normally distributed demand and another with distribution
free demand. For each approach, a Lagrange multiplier method is applied in order to solve
the discussed constrained inventory models and a solution procedure is developed to find
optimal values. This study, also, shows that the respective budget and storage space
constrained inventory models to be minimized are jointly convex in the decision variables.
Numerical examples are also presented to illustrate the models
Care Seeking Patterns of STIs-Associated Symptoms in Iran: Findings of a Population-Based Survey
Background: Understanding the prevalence of symptoms associated with sexually transmitted infections (STIs)
and how care is sought for those symptoms are important components of STIs control and prevention. People’s
preference between public and private service providers is another important part of developing a well-functioning
STIs surveillance system.
Methods:This cross-sectional survey was carried out in spring 2011, using a nonrandom quota sample of 1190
participants (52% female) in 4 densely-populated cities of Tehran, Kerman, Shiraz, and Babol. Two predictive
logistic regression models were constructed to assess the association between the socio-demographic determinants
(independent variables) and the dependent variables of history of STIs-associated symptom and seeking care.
Results:Around 57% (677 out of 1190; men: 29.70% and women: 81.80%) had experienced at least one STIsassociated symptom during the previous year. History of experiencing STIs-associated symptoms among men, was
negatively significantly associated with older age (adjusted odds ratio [AOR] = 0.34, CI 95%: 0.17-0.67). Women
who were married, in older ages, and had higher educations were more likely to report a recent (past year) STIs
symptom, however all were statistically insignificant in both bivariate and multivariable models. Among those
who have had STIs-associated symptoms in the last year, 31.15% did nothing to improve their symptoms, 8.03%
attempted self-treatment by over-the-counter (OTC) medications or traditional remedies, and 60.93% sought
care in health facilities. In both bivariate and multivariable analyses, care seeking among men was insignificantly
associated with any of the collected demographic variables. Care seeking among women was positively significantly
associated with being married (AOR = 2.48, 95% CI: 1.60-3.84).
Conclusion:The reported prevalence of STIs-associated symptoms among our participants is concerning. A
considerable number of participants had delayed seeking care and treatment or self-medicated. People should be
informed about their sexual health and the consequences of delaying or avoiding seeking care for STIs. Participants
preferred seeking care at private sectors which calls for engaging both public and private health sectors for reporting
and following up STIs cases
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