Mitigation of Anti-Drug Antibody Production for Augmenting Anticancer Efficacy of Therapeutic Protein via Co-Injection of Nano-Rapamycin.

The induction of anti-drug antibody (ADA) is a formidable challenge for protein-based therapy. Trichosanthin (TCS) as a class of ribosome-inactivating proteins is widely studied in tumor treatment. However, the immunogenicity can induce the formation of ADA, which can cause hypersensitivity reactions and neutralize the efficacy of TCS, thus limiting its clinical application in cancer therapy. Here, a promising solution to this issue is presented by co-administration of the rapamycin nanoparticles and TCS. PEGylated rapamycin amphiphilic molecule is designed and synthesized as a prodrug and a delivery carrier, which can self-assemble into a nanoparticle system with encapsulation of free rapamycin, a hydrophobic drug. It is found that co-injection of the PEGylated rapamycin nanoparticles and TCS could mitigate the formation of anti-TCS antibody via inducing durable immunological tolerance. Importantly, the combination of TCS and the rapamycin nanoparticles has an enhanced effect on inhibit the growth of breast cancer. This work provides a promising approach for protein toxin-based anticancer therapy and for promoting the clinical translation.

Investigation of Parameter Sensitivity and the Physical Mechanism for the Formation of a Core-Skin-Core (CSC) Structure in Two-Stage Co-Injection Molding.

One of the main challenges in co-injection molding is how to predict the skin to core morphology accurately and then manage it properly, especially after skin material has been broken through. In this study, the formation of the Core-Skin-Core (CSC) structure and its physical mechanism in a two-stage co-injection molding has been studied based on the ASTM D638 TYPE V system by using both numerical simulation and experimental observation. Results showed that when the skin to core ratio is selected properly (say 30/70), the CSC structure can be observed clearly at central location for 30SFPP/30SFPP system. When the skin to core ratio and operation conditions are fixed, regardless of material arrangement (including 30SFPP/30SFPP; PP/PP; 30SFPP/PP; and PP/30SFPP systems), the morphologies of the CSC structures are very close for all systems. This CSC structure can be further validated by using µ-CT scan and image analysis technologies perfectly. Furthermore, the influences of various operation parameters on the CSC structure variation have been investigated. Results exhibited that the CSC structure does not change significantly irrespective of the flow rate changing, melt temperature varying, or even mold temperature being modified. Moreover, the mechanism to generate the CSC structure can be derived using the melt front movement of the numerical simulation. It is worth noting that after the skin material was broken through, the core material travelled ahead with fountain flow to occupy the flow front. In the same period, the proper amount of skin material with certain inertia of enough kinetic energy will keep going to penetrate the new coming core material to travel until the end of filling. It ends up with this special CSC structure.

Efficient visual screening of CRISPR/Cas9 genome editing in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome editing has been applied to a wide variety of organisms, including nematodes such as Caenorhabditis elegans and Pristionchus pacificus. In these nematodes, genome editing is achieved by microinjection of Cas9 protein and guide RNA into the hermaphrodite gonads. However, P. pacificus is less efficient in CRISPR/Cas9 genome editing and exogenous gene expression. Therefore, it takes considerable time and effort to screen for target mutants if there are no visual markers that indicate successful injection. To overcome this problem, co-injection markers (gRNA for Ppa-prl-1, which induces the roller phenotype, and Ppa-egl-20p::turboRFP, a plasmid expressing a fluorescent protein) have been developed in P. pacificus. By selecting worms with the roller phenotype or turboRFP expression, screening efficiency is substantially increased to obtain worms with desired mutations. Here, we describe a step-by-step protocol for the visual screening system for CRISPR/Cas9 genome editing in P. pacificus. We also describe technical tips for microinjection, which is difficult for beginners. This protocol will facilitate genome editing in P. pacificus and may be applied to other nematode species.

Prevalence and correlates of simultaneous, multiple substance injection (co-injection) among people who inject drugs in Melbourne, Australia.

AIMS: To estimate the prevalence of and risk factors associated with concurrent injection of multiple substances (co-injection) among a community-recruited cohort of people who inject drugs. DESIGN: Cross-sectional study. SETTING: Melbourne, Australia. PARTICIPANTS: A sample of 720 actively injecting participants from the Melbourne Injecting Drug User Cohort Study (33% female) was extracted. MEASUREMENTS: We constructed two statistical models: a logistic regression model analysing correlates of co-injection of any substance combination in the past month and a multinomial logistic regression model analysing correlates of three mutually exclusive groups: heroin-diphenhydramine co-injection only, co-injection of other substances and no co-injection. Risk factors examined included drug use characteristics, demographic characteristics, health service use, hepatitis C status, injection risk behaviours and previous experience of non-fatal overdose. FINDINGS: One-third [n = 226, 31%; 95% confidence interval (CI): 28-34%] of participants reported co-injecting substances within the past month, with equal numbers of participants reporting injecting combinations of heroin-diphenhydramine (n = 121, 54%; 95% CI = 48-60%) and heroin-methamphetamine (n = 121, 54%; 95% CI = 48-60%). In logistic regression analyses, reporting co-injection of any substance combination was associated with male sex [adjusted odds ratio (aOR) = 1.80, 95% CI = 1.18-2.74, P = 0.006] and injecting daily or more frequently (aOR = 2.04, 95% CI = 1.31-3.18, P = 0.002). In multinomial logistic regression analyses, participants reporting heroin-diphenhydramine co-injection only were significantly more likely to report groin injecting [adjusted relative risk ratio (aRRR) = 6.16, 95% CI = 2.80-13.56, P < 0.001] and overdose (requiring an ambulance) in the past 12 months (aRRR = 2.81, 95% CI = 1.17-6.72, P = 0.021) compared with participants reporting no co-injection or co-injection of other substances. CONCLUSIONS: A substantial proportion of people who inject drugs report co-injection of multiple substances, which is associated with a range of socio-demographic, drug use and health service use risk factors.

Motivations for crystal methamphetamine-opioid co-injection/co-use amongst community-recruited people who inject drugs: a qualitative study.

AIMS: We report on motivations for crystal methamphetamine-opioid co-use/co-injection through narratives of people who inject drugs during a period of increased crystal methamphetamine use reporting in Australia. METHODS: Fourteen in-depth interviews were undertaken with selected participants (12 male, 2 female) from the Melbourne Injecting Drug User Cohort Study, including those in and out of opioid substitution therapy (OST). RESULTS: The main motivations for co-use reported by participants were as follows: (1) that heroin could be used to reduce the negative side effects of heavy crystal methamphetamine use, particularly during the 'comedown' phase; (2) that small quantities of crystal methamphetamine used with heroin could prolong the intoxication effect of heroin, and hence the time before opioid withdrawal; (3) that co-injection of crystal methamphetamine and heroin produced a more desirable intoxication effect than using either substance on its own and; (4) that crystal methamphetamine provided a substitute 'high' for heroin after commencing OST treatment. CONCLUSIONS: Co-use of methamphetamine and opioids has been used by people who inject drugs to facilitate intoxication, sometimes as the result of ineffective opioid substitution therapy (OST) treatment and perceived lack of pleasure after stabilisation on OST treatment.

Screening for CRISPR/Cas9-induced mutations using a co-injection marker in the nematode Pristionchus pacificus.

CRISPR/Cas9 genome-editing methods are used to reveal functions of genes and molecular mechanisms underlying biological processes in many species, including nematodes. In evolutionary biology, the nematode Pristionchus pacificus is a satellite model and has been used to understand interesting phenomena such as phenotypic plasticity and self-recognition. In P. pacificus, CRISPR/Cas9-mediated mutations are induced by microinjecting a guide RNA (gRNA) and Cas9 protein into the gonads. However, mutant screening is laborious and time-consuming due to the absence of visual markers. In this study, we established a Co-CRISPR strategy by using a dominant roller marker in P. pacificus. We found that heterozygous mutations in Ppa-prl-1 induced the roller phenotype, which can be used as an injection marker. After the co-injection of Ppa-prl-1 gRNA, target gRNA, and the Cas9 protein, roller progeny and their siblings were examined using the heteroduplex mobility assay and DNA sequencing. We found that some of the roller and non-roller siblings had mutations at the target site. We used varying Cas9 concentrations and found that a higher concentration of Cas9 did not increase genome-editing events. The Co-CRISPR strategy promotes the screening for genome-editing events and will facilitate the development of new genome-editing methods in P. pacificus.

Investigation on the Fiber Orientation Distributions and Their Influence on the Mechanical Property of the Co-Injection Molding Products.

In recent years, due to the rapid development of industrial lightweight technology, composite materials based on fiber reinforced plastics (FRP) have been widely used in the industry. However, the environmental impact of the FRPs is higher each year. To overcome this impact, co-injection molding could be one of the good solutions. But how to make the suitable control on the skin/core ratio and how to manage the glass fiber orientation features are still significant challenges. In this study, we have applied both computer-aided engineering (CAE) simulation and experimental methods to investigate the fiber feature in a co-injection system. Specifically, the fiber orientation distributions and their influence on the tensile properties for the single-shot and co-injection molding have been discovered. Results show that based on the 60:40 of skin/core ratio and same materials, the tensile properties of the co-injection system, including tensile stress and modulus, are a little weaker than that of the single-shot system. This is due to the overall fiber orientation tensor at flow direction (A11) of the co-injection system being lower than that of the single-shot system. Moreover, to discover and verify the influence of the fiber orientation features, the fiber orientation distributions (FOD) of both the co-injection and single-shot systems have been observed using micro-computerized tomography (µ-CT) technology to scan the internal structures. The scanned images were further utilizing Avizo software to perform image analyses to rebuild the fiber structure. Specifically, the fiber orientation tensor at flow direction (A11) of the co-injection system is about 89% of that of the single-shot system in the testing conditions. This is because the co-injection part has lower tensile properties. Furthermore, the difference of the fiber orientation tensor at flow direction (A11) between the co-injection and the single-shot systems is further verified based on the fiber morphology of the µ-CT scanned image. The observed result is consistent with that of the FOD estimation using µ-CT scan plus image analysis.

Tumor-Associated Macrophage Isolation and In Vivo Analysis of Their Tumor-Promoting Activity.

Characterization of individual cell populations from the tumor microenvironment is critical to understand their functional contribution to tumor progression. Magnetic bead enrichment and fluorescence-activated cell sorting (FACS) allow for the isolation of specific cell types that can be used in downstream applications, including in vitro and in vivo functional studies and molecular profiling. In this chapter, we describe the process of isolation of tumor-associated macrophages (TAMs) from primary murine breast tumors subsequent to therapeutic or experimental intervention. Additionally, we further detail how to analyze their ability to support tumor cell growth by co-injecting isolated TAMs with tumor cells orthotopically into the mammary gland of immune-deficient hosts, and monitoring tumor progression by live imaging and caliper measurement.

Identifying tumor promoting genomic alterations in tumor-associated fibroblasts via retrovirus-insertional mutagenesis.

Tumor-associated fibroblasts (TAFs) are often essential for solid tumor growth. However, few genetic or epigenetic alterations have been found in TAFs during the progression of solid tumors. Employing a tumor-stromal cell co-injection model, we adapted here retroviral-insertional mutagenesis to stromal cells to identify novel tumor-associated genes in TAFs. We successfully identified 20 gene candidates that might modulate tumor growth if altered in TAFs at genomic level. To validate our finding, the function of one of the candidate genes, tubulin tyrosine ligase (Ttl), was further studied in TAFs from fibrosarcoma, colon, breast and hepatocarcinoma. We demonstrated that down-regulated TTL expression in TAFs indeed promoted tumor growth in mice. Interestingly, decreased expression of TTL in tumor stromal cells also correlated with poor outcome in human colon carcinoma. Thus, the co-injection model of tumor cells with retrovirus-modified fibroblasts proved a valid method to identify tumor-modulating genes in TAFs, allowing for a deeper insight into the role of the stroma for tumor development.

Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose.

A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.

X-ray Microtomography of Intermittency in Multiphase Flow at Steady State Using a Differential Imaging Method.

We imaged the steady state flow of brine and decane in Bentheimer sandstone. We devised an experimental method based on differential imaging to examine how flow rate impacts impact the pore-scale distribution of fluids during coinjection. This allows us to elucidate flow regimes (connected, or breakup of the nonwetting phase pathways) for a range of fractional flows at two capillary numbers, Ca, namely 3.0 × 10-7 and 7.5 × 10-6. At the lower Ca, for a fixed fractional flow, the two phases appear to flow in connected unchanging subnetworks of the pore space, consistent with conventional theory. At the higher Ca, we observed that a significant fraction of the pore space contained sometimes oil and sometimes brine during the 1 h scan: this intermittent occupancy, which was interpreted as regions of the pore space that contained both fluid phases for some time, is necessary to explain the flow and dynamic connectivity of the oil phase; pathways of always oil-filled portions of the void space did not span the core. This phase was segmented from the differential image between the 30 wt % KI brine image and the scans taken at each fractional flow. Using the grey scale histogram distribution of the raw images, the oil proportion in the intermittent phase was calculated. The pressure drops at each fractional flow at low and high flow rates were measured by high-precision differential pressure sensors. The relative permeabilities and fractional flow obtained by our experiment at the mm-scale compare well with data from the literature on cm-scale samples.

MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B.

The interactions of the chimeric peptide MCRT (YPFPFRTic-NH2), based on morphiceptin and neuropeptide FF derivative PFRTic-NH2, on the effects of endokinin A/B (EKA/B) on mean arterial blood pressure of the urethane-anaesthetized rat have been investigated in the absence and presence of tachykinin receptor antagonists, naloxone and NO synthase inhibitors. While MCRT produced dose dependent decreases in mean arterial pressure, in its presence only a small but statistically insignificant decreases in the magnitude and the time course of the depressor effect of EKA/B (10nmol/kg) were observed. MCRT had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100nmol/kg). The tachykinin NK1 receptor antagonist SR140333B (1mg/kg) and the NK3 antagonist SR142891 (2.79mg/kg) both reduced the hypotensive effects of EKA/B and MCRT alone and blocked those of the two peptides in combination. The NK2 antagonist GR159897 (4mg/kg) partially blocked the depressor effects of EKA/B and MCRT alone. Naloxone (2mg/kg) completely blocked the depressor effect of MCTR, but partially blocked that of EKA/B. The NO synthase inhibitor l-NAME (50mg/kg) partially blocked the depressor effects of EKA/B, MCRT, and EKA/B + MCRT. These results could help to better understand the role of tachykinin receptors, opioid receptors and neuropeptide FF receptors in cardiovascular system.

Pain regulation of endokinin A/B or endokinin C/D on chimeric peptide MCRT in mice.

The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-NH2, based on YPFP-NH2 and PFRTic-NH2) at the supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, whereas 30 pmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhanced antinociception, which was nearly equal to the sum of the mathematical values of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly compared with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which may potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D, and MCRT and to better understand the complicated pain regulation of NK1 and µ opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different µ subtypes; and (2) picomolar EKA/B might motivate the endogenous NPFF system after NK1 activation.

Opposite Effects of Coinjection and Distant Injection of Mesenchymal Stem Cells on Breast Tumor Cell Growth.

UNLABELLED: : Mesenchymal stem cells (MSCs) usually promote tumor growth and metastasis. By using a breast tumor 4T1 cell-based animal model, this study determined that coinjection and distant injection of allogeneic bone marrow-derived MSCs with tumor cells could exert different effects on tumor growth. Whereas the coinjection of MSCs with 4T1 cells promoted tumor growth, surprisingly, the injection of MSCs at a site distant from the 4T1 cell inoculation site suppressed tumor growth. We further observed that, in the distant injection model, MSCs decreased the accumulation of myeloid-derived suppressor cells and regulatory T cells in tumor tissues by enhancing proinflammatory factors such as interferon-γ, tumor necrosis factor-α, Toll-like receptor (TLR)-3, and TLR-4, promoting host antitumor immunity and inhibiting tumor growth. Unlike previous reports, this is the first study reporting that MSCs may exert opposite roles on tumor growth in the same animal model by modulating the host immune system, which may shed light on the potential application of MSCs as vehicles for tumor therapy and other clinical applications. SIGNIFICANCE: Mesenchymal stem cells (MSCs) have been widely investigated for their potential roles in tissue engineering, autoimmune diseases, and tumor therapeutics. This study explored the impact of coinjection and distant injection of allogeneic bone marrow-derived MSCs on mouse 4T1 breast cancer cells. The results showed that the coinjection of MSCs and 4T1 cells promoted tumor growth. MSCs might act as the tumor stromal precursors and cause immunosuppression to protect tumor cells from immunosurveillance, which subsequently facilitated tumor metastasis. Interestingly, the distant injection of MSCs and 4T1 cells suppressed tumor growth. Together, the results of this study revealed the dual functions of MSCs in immunoregulation.

Enzyme-Controlled Intracellular Self-Assembly of (18)F Nanoparticles for Enhanced MicroPET Imaging of Tumor.

Herein, we report the development of a new "smart" radioactive probe (i.e., 1) which can undergo furin-controlled condensation and self-assembly of radioactive nanoparticles (i.e., 1-NPs) in tumor cells and its application for enhanced microPET imaging of tumors in nude mice co-injected with its cold analog (i.e., 1-Cold). Furin-controlled condensation of 1-Cold and self-assembly of its nanoparticles (i.e., 1-Cold-NPs) in vitro were validated and characterized with HPLC, mass spectra, SEM, and TEM analyses. Cell uptake studies showed that both 1 and 1-Cold have good cell permeability. TEM images of 1-Cold-treated MDA-MB-468 cells directly uncovered that the intracellular 1-Cold-NPs were at/near the location of furin (i.e., Golgi bodies). MTT results indicated that 50 µM 1-Cold did not impose cytotoxicity to MDA-MB-468 cells up to 12 hours. MicroPET imaging of MDA-MB-468 tumor-bearing mice indicated that mice co-injected with 1 and 1-Cold showed higher uptake and longer attenuation of the radioactivity in tumors than those mice only injected with same dosage of 1. Tumor uptake ratios of 1 between these two groups of mice reached the maximum of 8.2 folds at 240 min post injection. Biodistribution study indicated that the uptake ratios of 1 in kidneys between these two groups continuously increased and reached 81.9 folds at 240 min post injection, suggesting the formation of radioactive NPs (i.e., 1-NPs) in MDA-MB-468 tumors of mice co-injected with 1 and 1-Cold. And the nanoparticles were slowly digested and secreted from the tumors, accumulating in the kidneys. Our ''smart'' probe (i.e., 1), together with the strategy of co-injection, might help researchers trace the biomarkers of interest within a longer time window.

Interaction of endokinin A/B and (Mpa(6))-γ2-MSH-6-12 in pain regulation in mice.

The present study focused on the interactive effects of (Mpa(6))-γ2-MSH-6-12 (Mpa, spinal level) and endokinin A/B (EKA/B, supraspinal level) on pain regulation in mice. EKA/B (30 pmol) only weakened 100 pmol Mpa-induced hyperalgesia at 5 min, but could enhance it during 20-30 min. However, EKA/B (100 pmol) antagonized all dose levels of Mpa significantly at 5 min and blocked them completely at 10 min. EKA/B (3 nmol) co-injected with Mpa presented marked analgesia at 5 min and enduring hyperalgesia within 20-60 min. To investigate the underlying mechanisms between Mpa and EKA/B, SR140333B and SR142801 (NK1 and NK3 receptor antagonists, respectively) were utilized. SR140333B had no influence on Mpa, while SR142801 potentiated it during 20-30 min. Whereas, SR140333B and SR142801 could block the co-administration of Mpa and EKA/B (30 pmol) separately at 5 min and 30 min. These phenomena might attribute to that these two antagonists promoted the antagonism of EKA/B (30 pmol) at the early stage, while antagonized EKA/B preferentially in the latter period. SR140333B weakened the analgesia of EKA/B (3 nmol), but produced no effect on Mpa. However, SR140333B failed to affect the co-injection of Mpa and EKA/B, which implied that EKA/B cooperated with Mpa prior to SR140333B. These results could potentially help to better understand the interaction of NK and MrgC receptors in pain regulation in mice.

Steam and air co-injection in removing residual TCE in unsaturated layered sandy porous media.

Steam and air co-injection is a promising technique for volatile and semi-volatile organic contaminant remediation in heterogeneous porous media. In this study, removal of trichloroethene (TCE) with steam-air co-injection was investigated through a series of 2D sandbox experiments with different layered sand structures, and through numerical simulations. The results show that a layered structure with coarse sand, in which steam and air convection are relatively rapid, resulted in a higher removal rate and a larger removal ratio than those observed in an experiment using finer sand; however, the difference was not significant, and the removal ratios from three experiments ranged from 85% to 94%. Slight downward movement of TCE was observed for Experiment 1 (TCE initially in a fine sand zone encased in a coarse sand), while no such movement was observed for Experiment 2 (TCE initially in two fine sand layers encased in a coarse sand) or 3 (TCE initially in a silty sand zone encased in a coarse sand). Simulations show accumulation of TCE at the interface of the layered sands, which indicates a capillary barrier effect in restraining the downward movement of TCE. This effect is illustrated further by a numerical experiment with homogeneous coarse sand, in which continuous downward TCE movement to the bottom of the sandbox was simulated. Another numerical experiment with higher water saturation was also conducted. The results illustrate a complicated influence of water saturation on TCE removal in a layered sand structure.