Quantifying the impact of immunotherapy on RNA dynamics in cancer

BACKGROUND: Checkpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer types. Resolving intratumor transcriptomic changes induced by CPI may improve our understanding of the mechanisms of sensitivity and resistance. METHODS: We assembled a cohort of longitudinal pre-therapy and on-therapy samples from 174 patients treated with CPI across six cancer types by leveraging transcriptomic sequencing data from five studies. RESULTS: Meta-analyses of published RNA markers revealed an on-therapy pattern of immune reinvigoration in patients with breast cancer, which was not discernible pre-therapy, providing biological insight into the impact of CPI on the breast cancer immune microenvironment. We identified 98 breast cancer-specific correlates of CPI response, including 13 genes which are known IO targets, such as toll-like receptors TLR1, TLR4, and TLR8, that could hold potential as combination targets for patients with breast cancer receiving CPI treatment. Furthermore, we demonstrate that a subset of response genes identified in breast cancer are already highly expressed pre-therapy in melanoma, and additionally we establish divergent RNA dynamics between breast cancer and melanoma following CPI treatment, which may suggest distinct immune microenvironments between the two cancer types. CONCLUSIONS: Overall, delineating longitudinal RNA dynamics following CPI therapy sheds light on the mechanisms underlying diverging response trajectories, and identifies putative targets for combination therapy

Assisted evolution enables HIV-1 to overcome a high trim5α-imposed genetic barrier to rhesus macaque tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection

Thermal Properties of Carbon Nanotube–Copper Composites for Thermal Management Applications

Carbon nanotube–copper (CNT/Cu) composites have been successfully synthesized by means of a novel particles-compositing process followed by spark plasma sintering (SPS) technique. The thermal conductivity of the composites was measured by a laser flash technique and theoretical analyzed using an effective medium approach. The experimental results showed that the thermal conductivity unusually decreased after the incorporation of CNTs. Theoretical analyses revealed that the interfacial thermal resistance between the CNTs and the Cu matrix plays a crucial role in determining the thermal conductivity of bulk composites, and only small interfacial thermal resistance can induce a significant degradation in thermal conductivity for CNT/Cu composites. The influence of sintering condition on the thermal conductivity depended on the combined effects of multiple factors, i.e. porosity, CNTs distribution and CNT kinks or twists. The composites sintered at 600°C for 5 min under 50 MPa showed the maximum thermal conductivity. CNT/Cu composites are considered to be a promising material for thermal management applications

Combinatorial Computational Approaches to Identify Tetracycline Derivatives as Flavivirus Inhibitors

Limited structural information of drug targets, cellular toxicity possessed by lead compounds, and large amounts of potential leads are the major issues facing the design-oriented approach of discovering new leads. In an attempt to tackle these issues, we have developed a process of virtual screening based on the observation that conformational rearrangements of the dengue virus envelope protein are essential for the mediation of viral entry into host cells via membrane fusion. Screening was based solely on the structural information of the Dengue virus envelope protein and was focused on a target site that is presumably important for the conformational rearrangements necessary for viral entry. To circumvent the issue of lead compound toxicity, we performed screening based on molecular docking using structural databases of medical compounds. To enhance the identification of hits, we further categorized and selected candidates according to their novel structural characteristics. Finally, the selected candidates were subjected to a biological validation assay to assess inhibition of Dengue virus propagation in mammalian host cells using a plaque formation assay. Among the 10 compounds examined, rolitetracycline and doxycycline significantly inhibited plaque formation, demonstrating their inhibitory effect on dengue virus propagation. Both compounds were tetracycline derivatives with IC(50)s estimated to be 67.1 µM and 55.6 µM, respectively. Their docked conformations displayed common hydrophobic interactions with critical residues that affected membrane fusion during viral entry. These interactions will therefore position the tetracyclic ring moieties of both inhibitors to bind firmly to the target and, subsequently, disrupt conformational rearrangement and block viral entry. This process can be applied to other drug targets in which conformational rearrangement is critical to function

Tensor based multichannel reconstruction for breast tumours identification from DCE-MRIs

A new methodology based on tensor algebra that uses a higher order singular value decomposition to perform three-dimensional voxel reconstruction from a series of temporal images obtained using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is proposed. Principal component analysis (PCA) is used to robustly extract the spatial and temporal image features and simultaneously de-noise the datasets. Tumour segmentation on enhanced scaled (ES) images performed using a fuzzy C-means (FCM) cluster algorithm is compared with that achieved using the proposed tensorial framework. The proposed algorithm explores the correlations between spatial and temporal features in the tumours. The multi-channel reconstruction enables improved breast tumour identification through enhanced de-noising and improved intensity consistency. The reconstructed tumours have clear and continuous boundaries; furthermore the reconstruction shows better voxel clustering in tumour regions of interest. A more homogenous intensity distribution is also observed, enabling improved image contrast between tumours and background, especially in places where fatty tissue is imaged. The fidelity of reconstruction is further evaluated on the basis of five new qualitative metrics. Results confirm the superiority of the tensorial approach. The proposed reconstruction metrics should also find future applications in the assessment of other reconstruction algorithms

Diphenyl Difluoroketone: A Potent Chemotherapy Candidate for Human Hepatocellular Carcinoma

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was recently reported to inhibit proliferation of various cancer cells significantly. Here we try to determine the effect and mechanism of EF24 on hepatocellular carcinoma. 2 µM EF24 was found to inhibit the proliferation of PLC/PRF/5, Hep3B, HepG2, SK-HEP-1 and Huh 7 cell lines. However, even 8 µM EF24 treatment did not affect the proliferation of normal liver LO2 cells. Accordingly, 20 mg/kg/d EF24 inhibited the growth of the tumor xenografts conspicuously while causing no apparent change in liver, spleen or body weight. In addition, significant apoptosis and G2/M phase cell cycle arrest were found using flow cytometry. Besides, caspases and PARP activation and features typical of apoptosis including fragmented nuclei with condensed chromatin were also observed. Furthermore, the mechanism was targeted at the reduction of nuclear factor kappa b (NF-κB) pathway and the NF-κB–regulated gene products Bcl-2, COX-2, Cyclin B1. Our study has offered a strategy that EF24 being a therapeutic agent for hepatocellular carcinoma

Differential Gene Expression and Adherence of Escherichia coli O157:H7 In Vitro and in Ligated Pig Intestines

BACKGROUND: Escherichia coli O157:H7 strain 86-24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture. METHODOLOGY/PRINCIPAL FINDINGS: It was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO(3) (BHIN) plus lactose, pH was reduced to 5.5-5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA(15), TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA(48), Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels. CONCLUSIONS/SIGNIFICANCE: Presence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further

Activation of PI3K/mTOR pathway occurs in most adult low-grade gliomas and predicts patient survival

Recent evidence suggests the Akt-mTOR pathway may play a role in development of low-grade gliomas (LGG). We sought to evaluate whether activation of this pathway correlates with survival in LGG by examining expression patterns of proteins within this pathway. Forty-five LGG tumor specimens from newly diagnosed patients were analyzed for methylation of the putative 5′-promoter region of PTEN using methylation-specific PCR as well as phosphorylation of S6 and PRAS40 and expression of PTEN protein using immunohistochemistry. Relationships between molecular markers and overall survival (OS) were assessed using Kaplan-Meier methods and exact log-rank test. Correlation between molecular markers was determined using the Mann-Whitney U and Spearman Rank Correlation tests. Eight of the 26 patients with methylated PTEN died, as compared to 1 of 19 without methylation. There was a trend towards statistical significance, with PTEN methylated patients having decreased survival (P = 0.128). Eight of 29 patients that expressed phospho-S6 died, whereas all 9 patients lacking p-S6 expression were alive at last follow-up. There was an inverse relationship between expression of phospho-S6 and survival (P = 0.029). There was a trend towards decreased survival in patients expressing phospho-PRAS40 (P = 0.077). Analyses of relationships between molecular markers demonstrated a statistically significant positive correlation between expression of p-S6(235) and p-PRAS40 (P = 0.04); expression of p-S6(240) correlated positively with PTEN methylation (P = 0.04) and negatively with PTEN expression (P = 0.03). Survival of LGG patients correlates with phosphorylation of S6 protein. This relationship supports the use of selective mTOR inhibitors in the treatment of low grade glioma

Siglecg Limits the Size of B1a B Cell Lineage by Down-Regulating NFκB Activation

BACKGROUND: B1 B cells are believed to be a unique lineage with a distinct developmental pathway, function and activation requirement. How this lineage is genetically determined remained largely obscure. METHODS AND PRINCIPAL FINDINGS: Using the Siglecg-deficient mice with a knockin of green-fluorescent protein encoding sequence, we show here that, although the Siglecg gene is broadly expressed at high levels in all stages and/or lineages of B cells tested and at lower levels in other lineages, its deletion selectively expanded the B1a B cell lineages, including the frequency of the B1 cell progenitor in the bone marrow and the number of B1a cells in the peritoneal cavity, by postnatal expansion. The expansion of B1a B cells in the peritoneal correlated with enhanced activation of NFkappaB and was ablated by an IKK inhibitor. CONCLUSION AND SIGNIFICANCE: Our data revealed a critical role for Siglec G-NFkappaB pathway in regulating B1a B cell lineage. These data lead to a novel model of B1a lineage development that explains a large array of genetic data in this field

First Clinical Investigation of Cone Beam Computed Tomography and Deformable Registration for Adaptive Proton Therapy for Lung Cancer

PURPOSE: An adaptive proton therapy workflow using cone beam computed tomography (CBCT) is proposed. It consists of an online evaluation of a fast range-corrected dose distribution based on a virtual CT (vCT) scan. This can be followed by more accurate offline dose recalculation on the vCT scan, which can trigger a rescan CT (rCT) for replanning. METHODS AND MATERIALS: The workflow was tested retrospectively for 20 consecutive lung cancer patients. A diffeomorphic Morphon algorithm was used to generate the lung vCT by deforming the average planning CT onto the CBCT scan. An additional correction step was applied to account for anatomic modifications that cannot be modeled by deformation alone. A set of clinical indicators for replanning were generated according to the water equivalent thickness (WET) and dose statistics and compared with those obtained on the rCT scan. The fast dose approximation consisted of warping the initial planned dose onto the vCT scan according to the changes in WET. The potential under- and over-ranges were assessed as a variation in WET at the target's distal surface. RESULTS: The range-corrected dose from the vCT scan reproduced clinical indicators similar to those of the rCT scan. The workflow performed well under different clinical scenarios, including atelectasis, lung reinflation, and different types of tumor response. Between the vCT and rCT scans, we found a difference in the measured 95% percentile of the over-range distribution of 3.4 ± 2.7 mm. The limitations of the technique consisted of inherent uncertainties in deformable registration and the drawbacks of CBCT imaging. The correction step was adequate when gross errors occurred but could not recover subtle anatomic or density changes in tumors with complex topology. CONCLUSIONS: A proton therapy workflow based on CBCT provided clinical indicators similar to those using rCT for patients with lung cancer with considerable anatomic changes