28 research outputs found
Targeted Sequencing for Hereditary Breast and Ovarian Cancer in BRCA1/2-Negative Families Reveals Complex Genetic Architecture and Phenocopies
Approximately 20% of breast cancer cases are attributed to increased family risk, yet variation in BRCA1/2 can only explain 20%-25% of cases. Historically, only single gene or single variant testing were common in at-risk family members, and further sequencing studies were rarely offered after negative results. In this study, we applied an efficient and inexpensive targeted sequencing approach to provide molecular diagnoses in 245 human samples representing 134 BRCA mutation-negative (BRCAX) hereditary breast and ovarian cancer (HBOC) families recruited from 1973 to 2019 by Dr. Henry Lynch. Sequencing identified 391 variants, which were functionally annotated and ranked based on their predicted clinical impact. Known pathogenic CHEK2 breast cancer variants were identified in five BRCAX families in this study. While BRCAX was an inclusion criterion for this study, we still identified a pathogenic BRCA2 variant (p.Met192ValfsTer13) in one family. A portion of BRCAX families could be explained by other hereditary cancer syndromes that increase HBOC risk: Li-Fraumeni syndrome (gene: TP53) and Lynch syndrome (gene: MSH6). Interestingly, many families carried additional variants of undetermined significance (VOUSs) that may further modify phenotypes of syndromic family members. Ten families carried more than one potential VOUS, suggesting the presence of complex multi-variant families. Overall, nine BRCAX HBOC families in our study may be explained by known likely pathogenic/pathogenic variants, and six families carried potential VOUSs, which require further functional testing. To address this, we developed a functional assay where we successfully re-classified one family\u27s PMS2 VOUS as benign
Contrast-enhanced ultrasound for differentiating benign from malignant focal solid renal lesions in pediatric patients
Abstract The contrast-enhanced ultrasound (CEUS) has been mainly applied to adults to differentiate benign and malignant renal lesions, however, the characteristics of CEUS in pediatric has not been as well studied as in adults. In the present work, the eligible pediatric patients who underwent renal CEUS between March 2016 and February 2023 were retrospectively analyzed. It included 20 lesions (median diameter, 8.4 cm; range, 1.8–18.0 cm) from 20 patients (median age, 28.0 months; range, 3.0–212.0 months; 9 boys) in malignant group and 5 lesions (median diameter, 3.8 cm; range, 1.3–7.5 cm) from 5 patients (median age, 25.0 months; range, 0.7–216.0 months; 2 boys) in benign group. The diagnostic performance was assessed. Nonparametric and Chi-square tests were performed. With hyperenhancement plus wash-out, CEUS showed a sensitivity of 95.0% [95% confidence interval (CI): 75.1%, 99.9%], a specificity of 80.0% (CI: 28.4%, 99.5%), a positive predictive value of 95.0% (CI: 75.1%, 99.9%) and a negative predictive value of 80.0% (CI: 28.4%, 99.5%). It suggested that CEUS is a valuable technique for identifying between malignant and benign renal lesions in children
Enzyme/pH-triggered anticancer drug delivery of chondroitin sulfate modified doxorubicin nanocrystal
AbstractIn this paper, we developed a novel strategy of preparing doxorubicin (DOX) nanocrystal (NC) exerting spherical particles with a diameter of 102 nm, which experienced following coating of chondroitin sulphate derivative (CSOA) shell via electrostatic and hydrophobic interactions. Such multifunctional outerwear resulted in drug nanocapsules with high drug loading content up to 70% and high colloidal stability under physiological conditions. It exhibited accelerated drug release behaviour when dispersing in hyaluronidase (HAase) containing medium or incubated with cancer cells. CSOA/NCs were effectively taken up by cancer cells via CD44 receptor-mediated endocytosis, but were rarely internalised into normal fibroblasts. With the comparison of typical drug-loaded micelles system (DOX/PEG-PCL), CSOA/NCs showed greater inhibition to cancer cells due to the targeted and sensitive drug delivery
Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion
Prevalent
single cell RNA amplification and sequencing chemistries
mainly focus on polyadenylated RNAs in eukaryotic cells by using oligoÂ(dT)
primers for reverse transcription. We develop a new RNA amplification
method, “easier-seq”, to reverse transcribe and amplify
the total RNAs, both with and without polyadenylate tails, from a
single cell for transcriptome sequencing with high efficiency, reproducibility,
and accuracy. By distributing the reverse transcribed cDNA molecules
into 1.5 Ă— 10<sup>5</sup> aqueous droplets in oil, the cDNAs
are isothermally amplified using random primers in each of these 65-pL
reactors separately. This new method greatly improves the ease of
single-cell RNA sequencing by reducing the experimental steps. Meanwhile,
with less chance to induce errors, this method can easily maintain
the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent
method can be seamlessly applied to prokaryotic cell RNA sequencing
Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments
We
develop a novel single-cell-based platform through digital counting
of amplified genomic DNA fragments, named multifraction amplification
(mfA), to detect the copy number variations (CNVs) in a single cell.
Amplification is required to acquire genomic information from a single
cell, while introducing unavoidable bias. Unlike prevalent methods
that directly infer CNV profiles from the pattern of sequencing depth,
our mfA platform denatures and separates the DNA molecules from a
single cell into multiple fractions of a reaction mix before amplification.
By examining the sequencing result of each fraction for a specific
fragment and applying a segment-merge maximum likelihood algorithm
to the calculation of copy number, we digitize the sequencing-depth-based
CNV identification and thus provide a method that is less sensitive
to the amplification bias. In this paper, we demonstrate a mfA platform
through multiple displacement amplification (MDA) chemistry. When
performing the mfA platform, the noise of MDA is reduced; therefore,
the resolution of single-cell CNV identification can be improved to
100 kb. We can also determine the genomic region free of allelic drop-out
with mfA platform, which is impossible for conventional single-cell
amplification methods
The effect of π-Conjugation on the self-assembly of micelles and controlled cargo release
AbstractHere we presented a novel micelle self-assembled from amphiphiles with π-conjugated moieties (OEG-DPH). The π-conjugated structural integrity of the micelles enabled stable encapsulation of Nile Red (NR, model drug). The self-assembly behaviour of the amphiphiles and the release profile of NR loaded micelles were investigated. Spherical core-shell structured NR loaded micelles with low CMC of 57 μg/mL and the efficient intracellular delivery process was monitored. This research provided a way to fabricate stable polymeric micelles and develop a practical nanocarrier for therapeutics delivery
Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes
The next generation
sequencing (NGS) technologies have been rapidly
evolved and applied to various research fields, but they often suffer
from losing long-range information due to short library size and read
length. Here, we develop a simple, cost-efficient, and versatile NGS
library preparation method, called tagmentation on microbeads (TOM).
This method is capable of recovering long-range information through
tagmentation mediated by microbead-immobilized transposomes. Using
transposomes with DNA barcodes to identically label adjacent sequences
during tagmentation, we can restore inter-read connection of each
fragment from original DNA molecule by fragment-barcode linkage after
sequencing. In our proof-of-principle experiment, more than 4.5% of
the reads are linked with their adjacent reads, and the longest linkage
is over 1112 bp. We demonstrate TOM with eight barcodes, but the number
of barcodes can be scaled up by an ultrahigh complexity construction.
We also show this method has low amplification bias and effectively
fits the applications to identify copy number variations
Digital Polymerase Chain Reaction in an Array of Femtoliter Polydimethylsiloxane Microreactors
We developed a simple, compact microfluidic device to
perform high
dynamic-range digital polymerase chain reaction (dPCR) in an array
of isolated 36-femtoliter microreactors. The density of the microreactors
exceeded 20 000/mm<sup>2</sup>. This device, made from polydimethylsiloxane
(PDMS), allows the samples to be loaded into all microreactors simultaneously.
The microreactors are completely sealed through the deformation of
a PDMS membrane. The small volume of the microreactors ensures a compact
device with high reaction efficiency and low reagent and sample consumption.
Future potential applications of this platform include multicolor
dPCR and massively parallel dPCR for next generation sequencing library
preparation