Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents.

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

Effect of different retention doses of ultrasound-guided polidocanol chemical ablation for benign cystic-solid thyroid nodules.

OBJECTIVE: To assess the influence of varying retention doses of ultrasound-guided polidocanol chemical ablation for benign cystic-solid thyroid nodules. METHODS: A retrospective study was conducted from December 2019 to January 2022, including 78 patients with benign cystic-solid thyroid nodules, of which 31 received polidocanol chemical ablation alone, 23 received polidocanol chemical plus thermal ablation, and 24 received open surgery. Patients who received polidocanol chemical ablation were assigned into groups based on the retention dose of polidocanol: 0 %, 10 %, 20 %, 30 %, and 50 %. Follow-ups were done at 1, 3, 6, and 12 months postoperatively. The volume of the nodules, postoperative complications, and recurrence of the nodules were examined before treatment and during follow-up visits. RESULTS: Total operation time and intraoperative bleeding volume for patients who received ablation were substantially lower than those for patients who received open surgery (P < 0.001). Among patients in the polidocanol chemical ablation group, volume shrinkage rate of thyroid nodules in the 10 % retention dose group was significantly lower than that in the 0 % retention dose group at 1, 3, and 6 months postoperatively (P < 0.05). The 30 % retention dose group had the highest nodule shrinkage rate (98.46 ± 1.55 %) at 12 months postoperatively, which was significantly higher than that in the 50 % retention dose group (P < 0.05). Among patients in the polidocanol chemical and thermal ablation group, the volume shrinkage rate of thyroid nodules in the 10 % and 30 % retention dose groups at 1 month postoperatively was significantly lower than that in the 0 % retention dose group (P < 0.05). Although volume shrinkage rate in the 20 % retention dose group after thermal ablation was higher than that in the 0 % retention dose group, the difference was not statistically significant (P > 0.05). In terms of adverse reactions, the incidence of hoarseness and coughing was higher in the open surgery group than in the polidocanol chemical ablation and polidocanol chemical and thermal ablation groups, but there was no significant difference (P > 0.05). CONCLUSION: Chemical ablation with polidocanol was safe and effective for therapy of benign cystic-solid thyroid nodules, and the optimal retention dose may be between 20 % and 30 %. Patients with poor efficacy from chemical ablation alone can receive safe and effective treatment through thermal ablation.

26SCS-Loaded SilMA/Col Composite Sponge with Well-Arranged Layers Promotes Angiogenesis-Based Diabetic Wound Repair by Mediating Macrophage Inflammatory Response.

Diabetic wound healing is a significant clinical challenge because abnormal immune cells in the wound cause chronic inflammation and impair tissue regeneration. Therefore, regulating the behavior and function of macrophages may be conducive to improving treatment outcomes in diabetic wounds. Herein, sulfated chitosan (26SCS)-containing composite sponges (26SCS-SilMA/Col-330) with well-arranged layers and high porosity were constructed based on collagen and silk fibroin, aiming to induce an appropriate inflammatory response and promote angiogenesis. The results indicated that the ordered topological structure of composite sponges could trigger the pro-inflammatory response of Mφs in the early stage, and rapid release of 26SCS in the early and middle stages (within the concentration range of 1-3 mg/mL) induced a positive inflammatory response; initiated the pro-inflammatory reaction of Mφs within 3 days; shifted M1 Mφs to the M2 phenotype within 3-7 days; and significantly up-regulated the expression of two typical angiogenic growth factors, namely VEGF and PDGF-BB, on day 7, leading to rapid HUVEC migration and angiogenesis. In vivo data also demonstrated that on the 14th day after surgery, the 26SCS-SilMA/Col-330-implanted areas exhibited less inflammation, faster re-epithelialization, more abundant collagen deposition and a greater number of blood vessels in the skin tissue. The composite sponges with higher 26SCS contents (the (5.0) 26SCS-SilMA/Col-330 and the (7.5) 26SCS-SilMA/Col-330) could better orchestrate the phenotype and function of Mφs and facilitate wound healing. These findings highlight that the 26SCS-SilMA/Col-330 sponges developed in this work might have great potential as a novel dressing for the treatment of diabetic wounds.

PEG-Sheddable Nanodrug Remodels Tumor Microenvironment to Promote Effector T Cell Infiltration and Revise Their Exhaustion for Breast Cancer Immunotherapy.

Low infiltration of cytotoxic T lymphocytes and their exhaustion manifest the two concurrent main hurdles for achieving effective tumor immunotherapy of triple-negative breast cancer. It is found that Galectin-9 blockage can revise the exhaustion of effector T cells, meanwhile the repolarization of protumoral M2 tumor-associated macrophages (TAMs) into tumoricidal M1-like ones can recruit effector T cells infiltrating into tumor to boost immune responses. Herein, a sheddable PEG-decorated and M2-TAMs targeted nanodrug incorporating Signal Transducer and Activator of Transcription 6 inhibitor (AS) and anti-Galectin-9 antibody (aG-9) is prepared. The nanodrug responds to acidic tumor microenvironment (TME) with the shedding of PEG corona and the release of aG-9, exerting local blockade of PD-1/Galectin-9/TIM-3 interaction to augment effector T cells via exhaustion reversing. Synchronously, targeted repolarization of M2-TAMs into M1 phenotype by AS-loaded nanodrug is achieved, which promotes tumor infiltration of effector T cells and thus synergizes with aG-9 blockade to boost the therapeutic efficacy. Besides, the PEG-sheddable approach endows nanodrug with stealth ability to reduce immune-related adverse effects caused by AS and aG-9. This PEG sheddable nanodrug holds the potential to reverse the immunosuppressive TME and increase effector T cell infiltration, which dramatically enhances immunotherapy in highly malignant breast cancer.

Softness enhanced macrophage-mediated therapy of inhaled apoptotic-cell-inspired nanosystems for acute lung injury.

Engineered nanosystems offer a promising strategy for macrophage-targeted therapies for various diseases, and their physicochemical parameters including surface-active ligands, size and shape are widely investigated for improving their therapeutic efficacy. However, little is known about the synergistic effect of elasticity and surface-active ligands. Here, two kinds of anti-inflammatory N-acetylcysteine (NAC)-loaded macrophage-targeting apoptotic-cell-inspired phosphatidylserine (PS)-containing nano-liposomes (PSLipos) were constructed, which had similar size and morphology but different Young's modulus (E) (H, ~ 100 kPa > Emacrophage vs. L, ~ 2 kPa < Emacrophage). Interestingly, these PSLipos-NAC showed similar drug loading and encapsulation efficiency, and in vitro slow-release behavior of NAC, but modulus-dependent interactions with macrophages. Softer PSLipos-L-NAC could resist macrophage capture, but remarkably prolong their targeting effect period on macrophages via durable binding to macrophage surface, and subsequently more effectively suppress inflammatory response in macrophages and then hasten inflammatory lung epithelial cell wound healing. Especially, pulmonary administration of PSLipos-L-NAC could significantly reduce the inflammatory response of M1-like macrophages in lung tissue and promote lung injury repair in a bleomycin-induced acute lung injury (ALI) mouse model, providing a potential therapeutic approach for ALI. The results strongly suggest that softness may enhance ligand-directed macrophage-mediated therapeutic efficacy of nanosystems, which will shed new light on the design of engineered nanotherapeutics.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Human Embryonic Stem-Cell-Derived Exosomes Repress NLRP3 Inflammasome to Alleviate Pyroptosis in Nucleus Pulposus Cells by Transmitting miR-302c.

Recent studies have shown that the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is extensively activated in the process of intervertebral disc degeneration (IVDD), leading to the pyroptosis of nucleus pulposus cells (NPCs) and the exacerbation of the pathological development of the intervertebral disc (IVD). Exosomes derived from human embryonic stem cells (hESCs-exo) have shown great therapeutic potential in degenerative diseases. We hypothesized that hESCs-exo could alleviate IVDD by downregulating NLRP3. We measured the NLRP3 protein levels in different grades of IVDD and the effect of hESCs-exo on the H2O2-induced pyroptosis of NPCs. Our results indicate that the expression of NLRP3 was upregulated with the increase in IVD degeneration. hESCs-exo were able to reduce the H2O2-mediated pyroptosis of NPCs by downregulating the expression levels of NLRP3 inflammasome-related genes. Bioinformatics software predicted that miR-302c, an embryonic stem-cell-specific RNA, can inhibit NLRP3, thereby alleviating the pyroptosis of NPCs, and this was further verified by the overexpression of miR-302c in NPCs. In vivo experiments confirmed the above results in a rat caudal IVDD model. Our study demonstrates that hESCs-exo could inhibit excessive NPC pyroptosis by downregulating the NLRP3 inflammasome during IVDD, and miR-302c may play a key role in this process.

Dynamics of a colloid-in-tube host-guest system.

Originated from supramolecular chemistry, the host-guest concept is generalized and appreciated across the fields of enzyme catalysis, biological channel conduction, and carbon nanotube transport, to name a few. Despite the extensive study of host-guest thermodynamics, it is still a fundamental challenge to directly measure its dynamics in real-space and real-time. Herein we approach such dynamics by direct imaging and tracking in a colloid-in-tube system, where self-assembled tubes are the hosts and sphere particles are the guests. The key difference from a previously reported static 1D confinement is that the present tubes are thermally actuated and capable of translations and rotations. It is the host tube thermal motions that impose a number of anomalies to guest particle dynamics including broadened distribution perpendicular to the tube, enhanced diffusion parallel to the tube phenomenologically resembling the rapid flow in ion channels or carbon nanotubes, and induced long-range particle-particle attraction analogous to capillary condensation. This work in the colloidal system is of wide implications for host-guest systems that are naturally embedded in thermal fluctuations such as transmembrane ion channels and carbon nanotube arrays in a soft matrix.

Combination of radiofrequency ablation and percutaneous ethanol injection versus radiofrequency ablation alone for hepatocellular carcinoma: a systematic review and meta-analysis.

INTRODUCTION: Radiofrequency ablation and percutaneous ethanol injection are important treatment modalities for hepatocellular carcinoma patients; Whether a combination treatment yields, additional benefit still remains controversial. METHODS: A systematic review and meta-analysis was concluded. Randomized controlled trials published before January 1, 2022, from PubMed, EMBASE, Scopus, and CNKI were searched. Studies were excluded when patients received different ablative treatment or had serious liver dysfunction. The risk of bias assessment was evaluated using the Cochrane Collaboration's tool. RESULTS: Ten studies, encompassing 854 patients, with histologically proven HCC were finally analyzed. The results demonstrated that patients who received RFA-PEI had slightly improvements in 1-year overall survival (OS) [risk ratio (RR): 1.11; 95% confidence interval (CI): 1.03, 1.19, I2 = 10%], 2-year OS (RR: 1.25; 95% CI: 1.12, 1.40, I2 = 0%), 3-year OS (RR: 1.42; 95% CI: 1.11, 1.83, I2 = 38%), 1-year local recurrence-free (LRF) proportion (RR: 1.2; 95% CI: 1.01, 1.42, I2 = 61%), and complete tumor necrosis (CTN) (RR: 1.32; 95% CI: 1.14, 1.53, I2 = 45%). Nevertheless, common complications, such as fever, were found to be significant (RR: 1.78, 95% CI: 1.13, 2.80). CONCLUSION: Despite RFA-PEI appearing to be superior for HCC patients with a compensated liver in terms of OS, current evidence contained moderate to significant heterogeneity, and it was difficult to draw a definite conclusion regarding the therapeutic management in terms of LRF and CTN.

Rare Carbon-Bridged Citrinin Dimers from the Starfish-Derived Symbiotic Fungus Penicillium sp. GGF16-1-2.

Four novel, rare carbon-bridged citrinin dimers, namely dicitrinones G-J (1-4), and five known analogs (5-9) were isolated from the starfish-derived fungus Penicillium sp. GGF 16-1-2. Their structures were elucidated by extensive spectroscopic analysis and quantum chemical calculations. Compounds 1-9 exhibited strong antifungal activities against Colletotrichum gloeosporioides with LD50 values from 0.61 µg/mL to 16.14 µg/mL. Meanwhile, all compounds were evaluated for their cytotoxic activities against human pancreatic cancer BXPC-3 and PANC-1 cell lines; as a result, compound 1 showed more significant cytotoxicities than the positive control against both cell lines. In addition, based on the analyses of the protein-protein interaction (PPI) network and Western blot, 1 could induce apoptosis by activating caspase 3 proteins (CASP3).

Pyranodipyran Derivatives with Tyrosyl DNA Phosphodiesterase 1 Inhibitory Activities and Fluorescent Properties from Aspergillus sp. EGF 15-0-3.

Four new benzodipyran racemates, namely (±)-aspergiletals A-D (3-6), representing a rare pyrano[4,3-h]chromene scaffold were isolated together with eurotiumide G (1) and eurotiumide F (2) from the soft-coral-derived fungus Aspergillus sp. EGF 15-0-3. All the corresponding optically pure enantiomers were successfully separated by a chiral HPLC column. The structures and configurations of all the compounds were elucidated based on the combination of NMR and HRESIMS data, chiral separation, single-crystal X-ray diffraction, quantum chemical 13C NMR, and electronic circular dichroism calculations. Meanwhile, the structure of eurotiumide G was also revised. The TDP1 inhibitor activities and photophysical properties of the obtained compounds were evaluated. In the TDP1 inhibition assay, as a result of synergy between (+)-6 and (-)-6, (±)-6 displayed strong inhibitory activity to TDP1 with IC50 values of 6.50 ± 0.73 µM. All compounds had a large Stokes shift and could be utilized for elucidating the mode of bioactivities by fluorescence imaging.

γ-Aromatic Butenolides of Microbial Source - A Review of Their Structures, Biological Activities and Biosynthesis.

γ-Aromatic butenolides (γ-AB) are an important type of structures found in many bioactive microbial secondary metabolites (SMs). γ-AB refer to a group of natural products (NPs) containing five-membered (unsaturated) lactones with 3-phenyl and 4-benzyl substituents. Their wide-range biological activities have inspired pharmaceutical chemists to explore its biosynthesis mechanisms and design strategies to construct the γ-AB skeleton. Recently, there are a great deal of interesting research progress on the structures, biological activities and biosynthesis of γ-AB. This review will focus on these aspects and summarize the important achievements of γ-AB from 1975 to 2021.

A Novel Integrated Pharmacokinetic-Pharmacodynamic Model to Evaluate Combination Therapy and Determine In Vivo Synergism.

Understanding pharmacokinetic (PK)-pharmacodynamic (PD) relationships is essential in translational research. Existing PK-PD models for combination therapy lack consideration of quantitative contributions from individual drugs, whereas interaction factor is always assigned arbitrarily to one drug and overstretched for the determination of in vivo pharmacologic synergism. Herein, we report a novel generic PK-PD model for combination therapy by considering apparent contributions from individual drugs coadministered. Doxorubicin (Dox) and sorafenib (Sor) were used as model drugs whose PK data were obtained in mice and fit to two-compartment model. Xenograft tumor growth was biphasic in mice, and PD responses were described by three-compartment transit models. This PK-PD model revealed that Sor (contribution factor = 1.62) had much greater influence on overall tumor-growth inhibition than coadministered Dox (contribution factor = 0.644), which explains the mysterious clinical findings on remarkable benefits for patients with cancer when adding Sor to Dox treatment, whereas there were none when adding Dox to Sor therapy. Furthermore, the combination index method was integrated into this predictive PK-PD model for critical determination of in vivo pharmacologic synergism that cannot be correctly defined by the interaction factor in conventional models. In addition, this new PK-PD model was able to identify optimal dosage combination (e.g., doubling experimental Sor dose and reducing Dox dose by 50%) toward much greater degree of tumor-growth inhibition (>90%), which was consistent with stronger synergy (combination index = 0.298). These findings demonstrated the utilities of this new PK-PD model and reiterated the use of valid method for the assessment of in vivo synergism. SIGNIFICANCE STATEMENT: A novel pharmacokinetic (PK)-pharmacodynamic (PD) model was developed for the assessment of combination treatment by considering contributions from individual drugs, and combination index method was incorporated to critically define in vivo synergism. A greater contribution from sorafenib to tumor-growth inhibition than that of coadministered doxorubicin was identified, offering explanation for previously inexplicable clinical observations. This PK-PD model and strategy shall have broad applications to translational research on identifying optimal dosage combinations with stronger synergy toward improved therapeutic outcomes.

MicroRNA-1291-5p Sensitizes Pancreatic Carcinoma Cells to Arginine Deprivation and Chemotherapy through the Regulation of Arginolysis and Glycolysis.

Cancer cells are dysregulated and addicted to continuous supply and metabolism of nutritional glucose and amino acids (e.g., arginine) to drive the synthesis of critical macromolecules for uncontrolled growth. Recent studies have revealed that genome-derived microRNA (miRNA or miR)-1291-5p (miR-1291-5p or miR-1291) may modulate the expression of argininosuccinate synthase (ASS1) and glucose transporter protein type 1 (GLUT1). We also developed a novel approach to produce recombinant miR-1291 agents for research, which are distinguished from conventional chemo-engineered miRNA mimics. Herein, we firstly demonstrated that bioengineered miR-1291 agent was selectively processed to high levels of target miR-1291-5p in human pancreatic cancer (PC) cells. After the suppression of ASS1 protein levels, miR-1291 perturbed arginine homeostasis and preferably sensitized ASS1-abundant L3.3 cells to arginine deprivation therapy. In addition, miR-1291 treatment reduced the protein levels of GLUT1 in both AsPC-1 and PANC-1 cells, leading to a lower glucose uptake (deceased > 40%) and glycolysis capacity (reduced approximately 50%). As a result, miR-1291 largely improved cisplatin efficacy in the inhibition of PC cell viability. Our results demonstrated that miR-1291 was effective to sensitize PC cells to arginine deprivation treatment and chemotherapy through targeting ASS1- and GLUT1-mediated arginolysis and glycolysis, respectively, which may provide insights into understanding miRNA signaling underlying cancer cell metabolism and development of new strategies for the treatment of lethal PC. SIGNIFICANCE STATEMENT: Many anticancer drugs in clinical use and under investigation exert pharmacological effects or improve efficacy of coadministered medications by targeting cancer cell metabolism. Using new recombinant miR-1291 agent, we revealed that miR-1291 acts as a metabolism modulator in pancreatic carcinoma cells through the regulation of argininosuccinate synthase- and glucose transporter protein type 1-mediated arginolysis and glycolysis. Consequently, miR-1291 effectively enhanced the efficacy of arginine deprivation (pegylated arginine deiminase) and chemotherapy (cisplatin), offering new insights into development of rational combination therapies.

Pharmacokinetic and Pharmacodynamic Factors Contribute to Synergism between Let-7c-5p and 5-Fluorouracil in Inhibiting Hepatocellular Carcinoma Cell Viability.

Pharmacological interventions for hepatocellular carcinoma (HCC) are hindered by complex factors, and rational combination therapy may be developed to improve therapeutic outcomes. Very recently, we have identified a bioengineered microRNA let-7c-5p (or let-7c) agent as an effective inhibitor against HCC in vitro and in vivo. In this study, we sought to identify small-molecule drugs that may synergistically act with let-7c against HCC. Interestingly, we found that let-7c exhibited a strong synergism with 5-fluorouracil (5-FU) in the inhibition of HCC cell viability as manifested by average combination indices of 0.3 and 0.5 in Hep3B and Huh7 cells, respectively. By contrast, coadministration of let-7c with doxorubicin or sorafenib inhibited HCC cell viability with, rather surprisingly, no or minimal synergy. Further studies showed that protein levels of multidrug resistance-associated protein (MRP) ATP-binding cassette subfamily C member 5 (MRP5/ABCC5), a 5-FU efflux transporter, were reduced around 50% by let-7c in HCC cells. This led to a greater degree of intracellular accumulation of 5-FU in Huh7 cells as well as the second messenger cyclic adenosine monophosphate, an endogenous substrate of MRP5. Since 5-FU is an irreversible inhibitor of thymidylate synthetase (TS), we investigated the interactions of let-7c with 5-FU at pharmacodynamic level. Interestingly, our data revealed that let-7c significantly reduced TS protein levels in Huh7 cells, which was associated with the suppression of upstream transcriptional factors as well as other regulatory factors. Collectively, these results indicate that let-7c interacts with 5-FU at both pharmacokinetic and pharmacodynamic levels, and these findings shall offer insight into molecular mechanisms of synergistic drug combinations. SIGNIFICANCE STATEMENT: Combination therapy is a common strategy that generally involves pharmacodynamic interactions. After identifying a strong synergism between let-7c-5p and 5-fluorouracil (5-FU) against hepatocellular carcinoma cell viability, we reveal the involvement of both pharmacokinetic and pharmacodynamic mechanisms. In particular, let-7c enhances 5-FU exposure (via suppressing ABCC5/MRP5 expression) and cotargets thymidylate synthase with 5-FU (let-7c reduces protein expression, whereas 5-FU irreversibly inactivates enzyme). These findings provide insight into developing rational combination therapies based on pharmacological mechanisms.

Novel approaches for efficient in vivo fermentation production of noncoding RNAs.

Genome-derived noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), play an essential role in the control of target gene expression underlying various cellular processes, and dysregulation of ncRNAs is involved in the pathogenesis and progression of various diseases in virtually all species including humans. Understanding ncRNA biology has opened new avenues to develop novel RNA-based therapeutics. Presently, ncRNA research and drug development is dominated by the use of ncRNA mimics that are synthesized chemically in vitro and supplemented with extensive and various types of artificial modifications and thus may not necessarily recapitulate the properties of natural RNAs generated and folded in living cells in vivo. Therefore, there are growing interests in developing novel technologies for in vivo production of RNA molecules. The two most recent major breakthroughs in achieving an efficient, large-scale, and cost-effective fermentation production of recombinant or bioengineered RNAs (e.g., tens of milligrams from 1 L of bacterial culture) are (1) using stable RNA carriers and (2) direct overexpression in RNase III-deficient bacteria, while other approaches offer a low yield (e.g., nano- to microgram scales per liter). In this article, we highlight these novel microbial fermentation-based technologies that have shifted the paradigm to the production of true biological ncRNA molecules for research and development.

Diffusive Adhesives for Water-Rich Materials: Strong and Tunable Adhesion Beyond the Interface.

It is notoriously difficult to adhere water-rich materials, such as hydrogels and biological tissues. Existing adhesives usually suffer from weak and nonadjustable adhesion strength, in part because the contact between the adhesive and substrate is largely restrained to the adhesive/substrate interface. In this study, we have attempted to overcome this shortcoming by developing a class of diffusive adhesives (DAs) that can extend adhesion deep into the substrate to maximize the adhesive/substrate contact. The DAs consist of hydrogel matrices and preloaded water-soluble monomers and crosslinkers that can diffuse extensively into the water-rich substrates after adhesive/substrate contact. Polymerization and crosslinking of the monomers are then triggered leading to a bridging network that interpenetrates the DA and substrate skeletons and topologically binds them together. This kind of adhesion, in the absence of adhesive/substrate covalent bonding, is of high strength and toughness, comparable to those of the best-performing natural and artificial adhesives. More importantly, we can precisely tune the adhesion strength on demand by manipulating the diffusion profile. It is envisioned that the DA family could be extended to include a large pool of hydrogel matrices and monomers, and that they could be particularly useful in biological and medical applications.

Bioengineering of a single long noncoding RNA molecule that carries multiple small RNAs.

Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180 nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260 nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296 nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.

Rare Intrasellar Arachnoid Cyst Distinguishing From Other Benign Cystic Lesions and its Surgical Strategies.

The study reported a case of an intrasellar arachnoid cyst with visual disturbances as the main symptom. Arachnoid cyst is a common intracranial benign space-occupying lesion, but rarely seen in intrasellar region with less than 100 cases reported available in English language literature. Therefore, it is still controversial about the diagnosis and treatment of such patients. This article reviewed previous literature and discussed the differential diagnosis and surgical strategies of intrasellar arachnoid cyst in combination with our own case.