De novo individualized disease modules reveal the synthetic penetrance of genes and inform personalized treatment regimens

Current understandings of individual disease etiology and therapeutics are limited despite great need. To fill the gap, we propose a novel computational pipeline that collects potent disease gene cooperative pathways to envision individualized disease etiology and therapies. Our algorithm constructs individualized disease modules de novo, which enables us to elucidate the importance of mutated genes in specific patients and to understand the synthetic penetrance of these genes across patients. We reveal that importance of the notorious cancer drivers TP53 and PIK3CA fluctuate widely across breast cancers and peak in tumors with distinct numbers of mutations and that rarely mutated genes such as XPO1 and PLEKHA1 have high disease module importance in specific individuals. Furthermore, individualized module disruption enables us to devise customized singular and combinatorial target therapies that were highly varied across patients, showing the need for precision therapeutics pipelines. As the first analysis of de novo individualized disease modules, we illustrate the power of individualized disease modules for precision medicine by providing deep novel insights on the activity of diseased genes in individuals.

Pooled -omic data from patient samples have enabled construction of cellular interaction modules that provides a system-level understanding of disease etiology. This new conceptualization of disease has led to discoveries of previously unknown mechanisms and has significantly expanded opportunities for therapeutic targeting (Iborra-Egea et al. 2017; Sharma et al. 2018). Specifically, system and network science has pinpointed novel pharmacological targets and opportunities for drug repurposing or drug–drug synergies (Zhao and Iyengar 2012). Advancements owing to system biology have been even more pronounced in oncology. The reconstruction of complex cancer disease modules describes tumor biology at the system level, which is particularly important for such a polygenic and dynamic disease (Zielinski et al. 2017; Lin et al. 2019).Despite these recent advancements, a truly individualized system approach has yet to be applied to individualized medicine. Oncology patients in particular experience highly variable disease phenotypes and drug responses. The need for precision approaches in oncology has therefore been well established, with numerous scientists and clinicians calling for innovation (Aronson and Rehm 2015; Relling and Evans 2015; Carrasco-Ramiro et al. 2017; Werner et al. 2017). Patient-derived xenograft (PDX) models and clinical studies have highlighted the heterogeneity of tumor mechanistic properties and therapeutic responses (Chiron et al. 2014; Dagogo-Jack and Shaw 2018; Xu et al. 2019). Some of this variability can be captured with patient stratification through disease subtype classification or biomarker testing, but the majority of inter-patient variability remains unexplained (Dagogo-Jack and Shaw 2018). The lack of broadly applicable biomarkers indicates that unique system-level interactions are at play within single patients. System and network biology is poised to capture these phenomena well, but new theoretical frameworks and computational approaches must be implemented to make such precision network biology a reality.Existing methodologies extract disease modules (or “disease networks”), which are highly perturbed subnetworks of the larger cellular interactome where disease gene interactions occur (Menche et al. 2015). Previous approaches have attempted to detect and prioritize individualized cancer drivers, but these algorithms infer their individualized analyses from cohort-level disease modules (Bashashati et al. 2012; Cho et al. 2016; Reyna et al. 2018). For example, the LIONESS algorithm uses aggregate disease modules generated by existing approaches to linearly interpolate individual sample modules (Kuijjer et al. 2019). We hypothesize that although some individual patient disease activity is recapitulated in the cohort disease modules, there are additional unexplored interactions detectable only at the individual patient level, which dictate patient-specific mechanisms, phenotypes, and therapeutic responses. We additionally suspect that at the gene level, there are patient-specific variations in pathogenicity. This is because patients possess highly varied basal cellular environments and mutations (The Cancer Genome Atlas Network 2012; Dagogo-Jack and Shaw 2018). Given that current approaches rely heavily or exclusively on cohort-inferred disease modules, we suspect that inter-patient variability and precision have been underrepresented.Although practical, using features inferred across the cohort fail to capture patient individualized features by disregarding rare unique factors within a patient. A new approach is needed to truly infer individualized disease modules that accurately recapitulate individualized disease. In this study, we examined on the collective actions of mutated genes to try to understand individualized disease at a deeper level. We hypothesized that cohort disease modules are poorly representative of individualized disease and that new insights in precision medicine would reveal themselves once we zoomed in on individual patients. Thus, we set out, first, to create a robust pipeline for individualized disease module construction and, second, to use these disease modules to characterize individualize disease pathobiology and therapeutics.     

The efficacy of postoperative pain management with ketorolac for day case oral surgery

The efficacy of the non-steroidal anti-inflammatory analgesic, ketorolac (Toradol), was investigated in 52 day case patients undergoing removal of impacted third molar teeth under intravenous sedation and local analgesia. The study was double-blind, randomized and placebo-controlled. A single 30 mg dose of ketorolac was administered intravenously just prior to induction of sedation with midazolam. Ketorolac was well tolerated and provided good postoperative analgesia. It is suggested that ketorolac is a useful addition to the analgesic armamentarium and appropriately prescribed, provides good pain relief following day case oral surgery.     

Screening of chondrogenic factors with a real‐time fluorescence‐monitoring cell line ATDC5‐C2ER: Identification of sorting nexin 19 as a novel factor

Objective

To establish a cell culture system for noninvasive and real‐time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors.

Methods

The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP‐2), or transforming growth factor β (TGFβ) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene.

Results

We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5‐C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP‐2, or TGFβ, showing good correspondence to the chondrogenic markers. Screening using the ATDC5‐C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain‐of‐function and loss‐of‐function analyses revealed a potent chondrogenic activity of SNX19.

Conclusion

We established the ATDC5‐C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation.

     

Epidural haematoma after evacuation of contralateral subdural haematoma

Background Sequentially evolving intracranial bilateral haematomas, where the second haematoma develops after the surgical removal of the first one is rarely reported. Aim To report a patient who developed an epidural haematoma after evacuation of a contralateral subdural haematoma. Methods A 49-year-old male was admitted to our department after head injury. A brain computerized tomography (CT) scan revealed an acute subdural haematoma in the right temporal area which was evacuated. During his stay in the intensive care unit, he was submitted to intracranial pressure monitoring, which soon rose. Results A new CT scan showed an acute epidural haematoma in the contralateral parietal area that was also evacuated. Conclusions While rising intracranial pressure after the evacuation of a traumatic haematoma is usually attributed to brain oedema or recurrent haematoma at the craniotomy site, the development of a contralateral epidural haematoma requiring surgical treatment should not be overlooked.     

Phytochemical studies on Stemona burkillii prain: two new dihydrostemofoline alkaloids

Two new dihydrostemofoline alkaloids, 11(S),12(R)-dihydrostemofoline (3) and stemoburkilline (4), along with stemofoline (1) and 2'-hydroxystemofoline (2) have been isolated from a root extract of Stemona burkillii Prain. The structure and relative configuration of 3 have been determined via spectroscopic data and from comparison with synthetic 11(S),12(S)-dihydrostemofoline (5). The configuration of the exo-cyclic alkene group in 4 is tentively assigned as E on the basis of mechanistic considerations.     

Alleviation of capsular formations on silicone implants in rats using biomembrane-mimicking coatings

Despite their popular use in breast augmentation and reconstruction surgeries, the limited biocompatibility of silicone implants can induce severe side effects, including capsular contracture – an excessive foreign body reaction that forms a tight and hard fibrous capsule around the implant. This study examines the effects of using biomembrane-mimicking surface coatings to prevent capsular formations on silicone implants. The covalently attached biomembrane-mimicking polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), prevented nonspecific protein adsorption and fibroblast adhesion on the silicone surface. More importantly, in vivo capsule formations around PMPC-grafted silicone implants in rats were significantly thinner and exhibited lower collagen densities and more regular collagen alignments than bare silicone implants. The observed decrease in α-smooth muscle actin also supported the alleviation of capsular formations by the biomembrane-mimicking coating. Decreases in inflammation-related cells, myeloperoxidase and transforming growth factor-β resulted in reduced inflammation in the capsular tissue. The biomembrane-mimicking coatings used on these silicone implants demonstrate great potential for preventing capsular contracture and developing biocompatible materials for various biomedical applications.     

A Computer Reconstruction of the Entire Coronary Arterial Tree Based on Detailed Morphometric Data

A rigorous analysis of blood flow must be based on the branching pattern and vascular geometry of the full vascular circuit of interest. It is experimentally difficult to reconstruct the entire vascular circuit of any organ because of the enormity of the vessels. The objective of the present study was to develop a novel method for the reconstruction of the full coronary vascular tree from partial measurements. Our method includes the use of data on those parts of the tree that are measured to extrapolate the data on those parts that are missing. Specifically, a two-step approach was employed in the reconstruction of the entire coronary arterial tree down to the capillary level. Vessels > 40 μm were reconstructed from cast data while vessels < 40 μm were reconstructed from histological data. The cast data were reconstructed one-bifurcation at a time while histological data were reconstructed one-sub-tree at a time by “cutting” and “pasting” of data from measured to missing vessels. The reconstruction algorithm yielded a full arterial tree down to the first capillary bifurcation with 1.9, 2.04 and 1.15 million vessel segments for the right coronary artery (RCA), left anterior descending (LAD) and left circumflex (LCx) trees, respectively. The node-to-node connectivity along with the diameter and length of every vessel segment was determined. Once the full tree was reconstructed, we automated the assignment of order numbers, according to the diameter-defined Strahler system, to every vessel segment in the tree. Consequently, the diameters, lengths, number of vessels, segments-per-element ratio, connectivity and longitudinal matrices were determined for every order number. The present model establishes a morphological foundation for future analysis of blood flow in the coronary circulation.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.