Inferring cellular and molecular processes in single-cell data with non-negative matrix factorization using Python, R and GenePattern Notebook implementations of CoGAPS.

Non-negative matrix factorization (NMF) is an unsupervised learning method well suited to high-throughput biology. However, inferring biological processes from an NMF result still requires additional post hoc statistics and annotation for interpretation of learned features. Here, we introduce a suite of computational tools that implement NMF and provide methods for accurate and clear biological interpretation and analysis. A generalized discussion of NMF covering its benefits, limitations and open questions is followed by four procedures for the Bayesian NMF algorithm Coordinated Gene Activity across Pattern Subsets (CoGAPS). Each procedure will demonstrate NMF analysis to quantify cell state transitions in a public domain single-cell RNA-sequencing dataset. The first demonstrates PyCoGAPS, our new Python implementation that enhances runtime for large datasets, and the second allows its deployment in Docker. The third procedure steps through the same single-cell NMF analysis using our R CoGAPS interface. The fourth introduces a beginner-friendly CoGAPS platform using GenePattern Notebook, aimed at users with a working conceptual knowledge of data analysis but without a basic proficiency in the R or Python programming language. We also constructed a user-facing website to serve as a central repository for information and instructional materials about CoGAPS and its application programming interfaces. The expected timing to setup the packages and conduct a test run is around 15 min, and an additional 30 min to conduct analyses on a precomputed result. The expected runtime on the user's desired dataset can vary from hours to days depending on factors such as dataset size or input parameters.

Deep Learning Model for Static Ocular Torsion Detection Using Synthetically Generated Fundus Images.

Purpose: The objective of the study is to develop deep learning models using synthetic fundus images to assess the direction (intorsion versus extorsion) and amount (physiologic versus pathologic) of static ocular torsion. Static ocular torsion assessment is an important clinical tool for classifying vertical ocular misalignment; however, current methods are time-intensive with steep learning curves for frontline providers. Methods: We used a dataset (n = 276) of right eye fundus images. The disc-foveal angle was calculated using ImageJ to generate synthetic images via image rotation. Using synthetic datasets (n = 12,740 images per model) and transfer learning (the reuse of a pretrained deep learning model on a new task), we developed a binary classifier (intorsion versus extorsion) and a multiclass classifier (physiologic versus pathologic intorsion and extorsion). Model performance was evaluated on unseen synthetic and nonsynthetic data. Results: On the synthetic dataset, the binary classifier had an accuracy and area under the receiver operating characteristic curve (AUROC) of 0.92 and 0.98, respectively, whereas the multiclass classifier had an accuracy and AUROC of 0.77 and 0.94, respectively. The binary classifier generalized well on the nonsynthetic data (accuracy = 0.94; AUROC = 1.00). Conclusions: The direction of static ocular torsion can be detected from synthetic fundus images using deep learning methods, which is key to differentiate between vestibular misalignment (skew deviation) and ocular muscle misalignment (superior oblique palsies). Translational Relevance: Given the robust performance of our models on real fundus images, similar strategies can be adopted for deep learning research in rare neuro-ophthalmologic diseases with limited datasets.

Adequacy of samples obtained via percutaneous core-needle rebiopsy for EGFR T790M molecular analysis in patients with non-small cell lung cancer following acquired resistance to first-line therapy: A systematic review and meta-analysis.

MICRO ABSTRACT: Rebiopsies characterizing resistance mutations in patients with non-small cell lung cancer (NSCLC) can guide personalized medicine and improve overall survival rates. In this systematic review, we examine the suitability of percutaneous core-needle biopsy (PT-CNB) to obtain adequate samples for molecular characterization of the acquired resistance mutation T790M. This review provides evidence that PT-CNB can obtain samples with high adequacy, with a mutation detection rate that is in accordance with prior literature. BACKGROUND: Non-small cell lung cancer (NSCLC) comprises 85% of all lung cancers and has seen improved survival rates with the rise of personalized medicine. Resistance mutations to first-line therapies, such as T790M, however, render first-line therapies ineffective. Rebiopsies characterizing resistance mutations inform therapeutic decisions, which result in prolonged survival. Given the high efficacy of percutaneous core-needle biopsy (PT-CNB), we conducted the first systematic review to analyze the ability of PT-CNB to obtain samples of high adequacy in order to characterize the acquired resistance mutation T790M in patients with NSCLC. METHODS: We performed a comprehensive literature search across PubMed, Embase, and CENTRAL. Search terms related to "NSCLC," "rebiopsy," and "PT-CNB" were used to obtain results. We included all prospective and retrospective studies that satisfied our inclusion and exclusion criteria. A random effects model was utilized to pool adequacy and detection rates of the chosen articles. We performed a systematic review, meta-analysis, and meta-regression to investigate the adequacy and T790M detection rates of samples obtained via PT-CNB. RESULTS: Out of the 173 studies initially identified, 5 studies met the inclusion and exclusion criteria and were chosen for our final cohort of 436 patients for meta-analysis. The pooled adequacy rate of samples obtained via PT-CNB was 86.92% (95% CI: [79.31%, 92.0%]) and the pooled T790M detection rate was 46.0% (95% CI: [26.6%, 66.7%]). There was considerable heterogeneity among studies (I2 > 50%) in both adequacy and T790M detection rates. CONCLUSION: PT-CNB can obtain adequate samples for T790M molecular characterization in NSCLC lung cancer patients. Additional prospective studies are needed to corroborate the results in this review.

Maintaining Contour with a Three-dimensional Interstitial Tissue Marker in 134 Lumpectomies.

Breast-conserving surgery (BCS) is meant to preserve the natural appearance of the breast; however, tissue volume deficits cannot always be compensated by soft tissue mobilization. A three-dimensional (3D) interstitial tissue marker (BioZorb) was designed to delineate the lumpectomy cavity for targeting boost irradiation, but an unexpected secondary benefit may be in guiding wound contraction and restoring contour to the lumpectomy bed. We analyze tissue volume excised at the time of lumpectomy as a function of device size selected. METHODS: In total, 134 consecutive lumpectomy patients implanted with BioZorb between May 2015 and February 2020 were retrospectively analyzed for tissue volume excised, device size used, location, and re-operation rates, including explantation of the device. RESULTS: An estimated 113 patients underwent device implantation at initial lumpectomy, and 21 at margin re-excision. Twenty-seven patients underwent re-excision, while 14 elected mastectomy for positive margins following insertion; 22 had the same device reimplanted. Mean lumpectomy volume was 79.0 cm3 (range 10.3-275.8 cm3) during the first implant procedure. Large-volume lumpectomies, averaging 136.5 cm3, were associated with selection of larger devices, which aided in restoring volume and maintaining breast contour. Three (2.2%) patients requested removal of the device. CONCLUSIONS: BioZorb implantation can be a safe and useful oncoplastic technique for restoring volume with BCS. Large-volume lumpectomies can be performed without contouring defects using the device. An unexpected secondary benefit of the device may be scaffolding for wound contraction.

#PrEP4Love: success and stigma following release of the first sex-positive PrEP public health campaign.

Pre-exposure prophylaxis (PrEP) is an effective yet under-utilised method for preventing HIV transmission in high-risk groups. Despite ongoing social marketing to increase PrEP awareness, few studies have evaluated public responses. This paper contextualises negative responses to Chicago's PrEP4Love campaign. In February 2016, a sex-positive ad campaign called PrEP4Love was launched online and throughout public spaces in Chicago. A gender and sexuality inclusive campaign, PrEP4Love is intended to be culturally responsive and sex positive, while retaining a focus on risk reduction. Advertisements prominently feature Black sexual minority men, and Black transgender women, and were strategically placed in diverse Chicago neighbourhoods. In response, there were 212 new callers to the PrEPLine during the two-month study period. Negative responses were concerned with: negatively depicting Black homosexuality (4), general anti-LGBTQ comments (7), adverse effects on children (6), sexually explicit nature (5), and general stigmatisation of racial minorities (4). Discussion focuses on sex-positive frameworks, normalising intimacy, stigma and historical mistrust of medical and pharmaceutical institutions, and the social meanings of biomedical prevention technologies (e.g. PrEP) in relation to dominant norms of sexuality and gender. This study is the first to investigate public responses to a sex-positive PrEP campaign. More studies of PrEP social marketing are needed to evaluate targeted public health campaigns to guide future PrEP promotion strategies.

Immunohistochemical and ultrastructural analysis of the maturing larval zebrafish enteric nervous system reveals the formation of a neuropil pattern.

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut. By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

Zinc Metallochaperones Reactivate Mutant p53 Using an ON/OFF Switch Mechanism: A New Paradigm in Cancer Therapeutics.

Purpose: Zinc metallochaperones (ZMC) are a new class of anticancer drugs that reactivate zinc-deficient mutant p53 by raising and buffering intracellular zinc levels sufficiently to restore zinc binding. In vitro pharmacodynamics of ZMCs indicate that p53-mutant activity is ON by 4-6 hours and is OFF by 24. We sought to understand the mechanism of this regulation and to translate these findings preclinically. We further sought to innovate the formulation of ZMCs to improve efficacy.Experimental Design: We performed in vitro mechanistic studies to determine the role of cellular zinc homeostatic mechanisms in the transient pharmacodynamics of ZMCs. We conducted preclinical pharmacokinetic, pharmacodynamic, and efficacy studies using a genetically engineered murine pancreatic cancer model (KPC) to translate these mechanistic findings and investigate a novel ZMC formulation.Results:In vitro, cellular zinc homeostatic mechanisms that restore zinc to its physiologic levels function as the OFF switch in ZMC pharmacodynamics. In vivo pharmacokinetic studies indicate that ZMCs have a short half-life (< 30 minutes), which is sufficient to significantly improve survival in mice expressing a zinc-deficient allele (p53R172H) while having no effect in mice expressing a non-zinc-deficient allele (p53R270H). We synthesized a novel formulation of the drug in complex with zinc and demonstrate this significantly improves survival over ZMC1.Conclusions: Cellular zinc homeostatic mechanisms function as an OFF switch in ZMC pharmacodynamics, indicating that a brief period of p53-mutant reactivation is sufficient for on-target efficacy. ZMCs synthesized in complex with zinc are an improved formulation. Clin Cancer Res; 24(18); 4505-17. ©2018 AACR.

U1 Adaptors Suppress the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts.

Targeting KRAS and MYC has been a tremendous challenge in cancer drug development. Genetic studies in mouse models have validated the efficacy of silencing expression of both KRAS and MYC in mutant KRAS-driven tumors. We investigated the therapeutic potential of a new oligonucleotide-mediated gene silencing technology (U1 Adaptor) targeting KRAS and MYC in pancreatic cancer. Nanoparticles in complex with anti-KRAS U1 Adaptors (U1-KRAS) showed remarkable inhibition of KRAS in different human pancreatic cancer cell lines in vitro and in vivo As a nanoparticle-free approach is far easier to develop into a drug, we refined the formulation of U1 Adaptors by conjugating them to tumor-targeting peptides (iRGD and cRGD). Peptides coupled to fluorescently tagged U1 Adaptors showed selective tumor localization in vivo Efficacy experiments in pancreatic cancer xenograft models showed highly potent (>90%) antitumor activity of both iRGD and (cRGD)2-KRAS Adaptors. U1 Adaptors targeting MYC inhibited pancreatic cancer cell proliferation caused by apoptosis in vitro (40%-70%) and tumor regressions in vivo Comparison of iRGD-conjugated U1 KRAS and U1 MYC Adaptors in vivo revealed a significantly greater degree of cleaved caspase-3 staining and decreased Ki67 staining as compared with controls. There was no significant difference in efficacy between the U1 KRAS and U1 MYC Adaptor groups. Our results validate the value in targeting both KRAS and MYC in pancreatic cancer therapeutics and provide evidence that the U1 Adaptor technology can be successfully translated using a nanoparticle-free delivery system to target two undruggable genes in cancer. Mol Cancer Ther; 16(8); 1445-55. ©2017 AACR.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

Breast reconstruction after mastectomy: A ten-year analysis of trends and immediate postoperative outcomes.

The landscape of breast reconstruction has changed significantly. This study assesses trends in type of reconstruction performed after mastectomy and impact on immediate postoperative complications. METHODS: Data for 67,450 patients undergoing mastectomy for breast cancer were analyzed using the National Surgical Quality Improvement Program (NSQIP) database for years 2005-2014. Primary outcomes were wound, nonwound related infections, and bleeding complications. Data were analyzed by univariate and multivariate analysis. RESULTS: The percentage of patients that underwent reconstruction after breast cancer increased from 26.94% in 2005 to 43.30% in 2014 (p < 0.01). There was increased wound (5.59%), bleeding (6.82%), and infection (1.80%) complications after flap-based reconstruction (p < 0.01). There was no difference in wound, infection, and bleeding complications between immediate implant reconstruction and tissue expander (TE) at 4.38 vs. 3.89% (p = 0.18), 0.82 vs. 0.7%, p = 0.46), and 0.76 vs. 0.64% (p = 0.45), respectively. Several independent factors were associated with increased wound complications in patients undergoing all or any forms of reconstruction after mastectomy such as being overweight (OR 1.38, CI 1.23-1.55), obese (OR 2.11, CI 1.89-2.35), morbidly obese (OR 3.84, CI 3.34-4.43), ASA Class III (OR 1.35, CI 1.08-1.69), ASA Class IV (OR 1.49, 1.06-2.10), diabetic (OR 1.28 , CI 1.14-1.43), and smokers (OR 1.76, CI 1.59-1.94). TRAM flap was associated with increased risk of wound complication (OR 1.87, CI 1.28-2.75). CONCLUSION: More women are undergoing reconstruction as utilization of TE increases drastically. Immediate implant placement has only seen moderate increase likely due to surgeon preference.

Outcomes of microwave ablation for colorectal cancer liver metastases: a single center experience.

BACKGROUND AND OBJECTIVES: Surgical management of colorectal cancer liver metastases continues to evolve to optimize oncologic outcomes while maximizing parenchymal preservation. Long-term data after intraoperative microwave ablation are limited. This study investigates outcomes and patterns of recurrence in patients who underwent intraoperative microwave ablation. METHODS: A retrospective analysis of 33 patients who underwent intraoperative microwave ablation of colorectal cancer liver metastases from 2009 to 2013 at our institution was performed. Perioperative and long-term data were reviewed to determine outcomes and patterns of recurrence. RESULTS: A total of 49 tumors were treated, ranging 0.5-5.5 cm in size. Median Clavien-Dindo classification was one. Median follow-up was 531 days, with 13 (39.4%) patients presenting with a recurrence. Median time to first recurrence was 364 days. In those patients, 1 (7.8%) presented with an isolated local recurrence in the liver. Only 1 of 7 ablated tumors greater than 3 cm recurred (14.3%). Overall survival was 35.2% at 4 years, with a 19.3% disease-free survival at 3.5 years. No perioperative variables predicted systemic or local recurrence. CONCLUSION: Intraoperative microwave ablation is a safe and effective modality for use in the treatment of colorectal cancer liver metastases in tumors as large as 5.5 cm in size.