Exchange of Proteins in Liposomes through Streptolysin O Pores.

Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in ∼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors.

[Radiation-Induced Undifferentiated Pleomorphic Sarcoma after Breast-Conserving Surgery-A Case Report].

The patient was a 73-year-old woman who had undergone breast-conserving surgery followed by irradiation (50 Gy/25 Fr)to the residual breast for left breast cancer 4 years before. Computed tomography for routine examination revealed a soft tissue mass on her left chest wall. Ultrasonography showed a hypoechoic mass with heterogeneous internal echo, 3.5×3.0×1.5 cm in size. Core-needle biopsy was performed, and histological examination revealed proliferation of spindle-shaped or pleomorphic and highly atypical cells. On immunohistochemistry, the tumor was negative for AE1/AE3, CD34, SMA, desmin, and S-100 and focally positive for CD68. Based on these findings, undifferentiated sarcoma was suspected. The patient underwent wide local excision of the chest wall with a surgical margin of 3-4 cm from the tumor edge. The histological diagnosis was undifferentiated pleomorphic sarcoma. Judging from the clinical course, this tumor was radiation-induced sarcoma. The patient remains disease-free 54 months after the operation.

[A Case of Effective Endovascular Treatment for Recurrent Bleeding from Advanced Breast Cancer].

A-58-year-old woman was diagnosed with breast cancer 8 years ago at another hospital, but refused surgical treatment. From 2 years ago, her skin invasion of cancer lesions began bleeding. The patient required frequent blood transfusions due to anemia associated with repeated bleeding. She was referred to our department for local treatment and palliative care. Diagnostic imaging revealed multiple lung, bone and liver metastasis. The patient refused to receive systemic chemotherapy, and she was recommended radiation therapy for repeated massive bleeding, but her consent was not obtained. She agreed to receive arterial embolization from the tumor-bearing vessels plus intravenous anti-cancer drug therapy. The hemostatic effect was observed for 4 to 5 weeks per treatment, and tumor reduction was also observed. She received a total of 6 treatments during 8 months until her death. These treatments were effective in maintaining quality of life at the end of life.

Sustainable proliferation of liposomes compatible with inner RNA replication.

Although challenging, the construction of a life-like compartment via a bottom-up approach can increase our understanding of life and protocells. The sustainable replication of genome information and the proliferation of phospholipid vesicles are requisites for reconstituting cell growth. However, although the replication of DNA or RNA has been developed in phospholipid vesicles, the sustainable proliferation of phospholipid vesicles has remained difficult to achieve. Here, we demonstrate the sustainable proliferation of liposomes that replicate RNA within them. Nutrients for RNA replication and membranes for liposome proliferation were combined by using a modified freeze-thaw technique. These liposomes showed fusion and fission compatible with RNA replication and distribution to daughter liposomes. The RNAs in daughter liposomes were repeatedly used as templates in the next RNA replication and were distributed to granddaughter liposomes. Liposome proliferation was achieved by 10 cycles of iterative culture operation. Therefore, we propose the use of culturable liposomes as an advanced protocell model with the implication that the concurrent supplement of both the membrane material and the nutrients of inner reactions might have enabled protocells to grow sustainably.

Flow Cytometric Analysis To Evaluate Morphological Changes in Giant Liposomes As Observed in Electrofusion Experiments.

Liposome fusion is a way of supplying additional components for in-liposome biochemical reactions. Electrofusion is a method that does not require the addition of fusogens, which often alter the liposome dispersion, and is therefore useful for repetitive liposome fusion. However, the details of electrofusion have not been elucidated because of the limitations surrounding observing liposomes using a microscope. Therefore, we introduced fluorescent markers and high-throughput flow cytometry to analyze the morphological changes that occur in liposome electrofusion. (i) The content mixing was evaluated by a calcein-Co2+-EDTA system, in which green fluorescence from dequenched free calcein is detected when the quenched calcein-Co2+ complex and EDTA are mixed together. (ii) Liposome destruction was evaluated from the decrease in the total membrane volume of giant liposomes. (iii) Liposome fission was evaluated from the increase in the number of giant liposomes. By applying the flow cytometric analysis, we investigated the effect of three parameters (DC pulse, AC field, and lipid composition) on liposome electrofusion. The larger numbers or higher voltages of DC pulses induced liposome fusion and destruction with higher probability. The longer application time of the AC field induced liposome fusion, fission, and destruction with higher probability. Higher content of negatively charged POPG (≥19%) strongly inhibited liposome electrofusion.

Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qß replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of ß-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

In vitro evolution of α-hemolysin using a liposome display.

In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of α-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained α-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.

The evolutionary enhancement of genotype-phenotype linkages in the presence of multiple copies of genetic material.

Genetic evolutionary mechanisms employed by protolife developed without accompanying regulatory mechanisms for the amounts of genetic material in protocells. When many copies of genetic material are present, inactive copies generated by mutations are not effectively excluded through phenotypic selection. We demonstrate a model of gene evolution initiated with different amounts of DNA inside artificial protocells. We adopted transcription- and translation-coupled RNA replication and liposome-based in vitro compartmentalization. Despite the fact that the average number of DNA copies in each liposome was 6.4, DNA encoding active genes was maintained until the 16th selection round. Our experimental and theoretical results indicated that gene evolution can occur in the presence of multiple DNA copies. Most genetic material became junk code through gene mutations, and consequently the linkage between genotype and phenotype was enhanced through the associated decreases in active genetic material.

[A case of S-1/CDDP-resistant recurrent gastric cancer responsive to capecitabine/CDDP].

We present a case of recurrent gastric cancer in which stable disease status was achieved for four months due to treatment with capecitabine/cisplatin (CDDP)after the failure of multiple anticancer drugs including S-1/CDDP. A 67-year-old man was diagnosed with multiple liver metastases one year after distal gastrectomy+D2 dissection for gastric cancer. S-1/CDDP was given as the first-line treatment, followed by paclitaxel (PTX), irinotecan (CPT-11), and docetaxel (DOC). The tumor in the anterior segment of the liver was resistant to all of these chemotherapies except for PTX, which is why the regimens were changed. However, this tumor shrank and achieved stable disease status for four months after capecitabine/CDDP therapy given as fifth-line treatment. Our case suggests that S-1 and capecitabine do not always exhibit cross-resistance. Therefore, capecitabine may be effective in S-1-pretreated patients, and vice versa.

Construction of a gene screening system using giant unilamellar liposomes and a fluorescence-activated cell sorter.

We have constructed a gene screening system composed of an in vitro transcription-translation system encapsulated within giant unilamellar liposomes and a fluorescence-activated cell sorter (FACS), which allows high-throughput screening of genes encoding proteins of interest. A mock gene library of ß-glucuronidase (GUS) was compartmentalized into liposomes at the single-molecule level, and liposomes exhibiting green fluorescence derived from hydrolysis of the fluorogenic substrate by the synthesized enzyme were sorted using FACS. More than 10-fold enrichment of GUS gene with higher catalytic activity was obtained when a single copy of the GUS gene was encapsulated in each liposome. Quantitative analysis of the enrichment factors and their liposome size dependencies showed that experimentally obtained and theoretical values were in agreement. Using this method, genes encoding active GUS were then enriched from a gene library of randomly mutated GUS genes. Only three rounds of screening were required, which was also consistent with our theoretical estimation.

Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry.

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.

Programmed vesicle fusion triggers gene expression.

The membrane properties of phospholipid vesicles can be manipulated to both regulate and initiate encapsulated biochemical reactions and networks. We present evidence for the inhibition and activation of reactions encapsulated in vesicles by the exogenous addition of charged amphiphiles. While the incorporation of cationic amphiphile exerts an inhibitory effect, complementation of additional anionic amphiphiles revitalize the reaction. We demonstrated both the simple hydrolysis reaction of ß-glucuronidase and the in vitro gene expression of this enzyme from a DNA template. Furthermore, we show that two vesicle populations decorated separately with positive and negative amphiphiles can fuse selectively to supply feeding components to initiate encapsulated reactions. This mechanism could be one of the rudimentary but effective means to regulate and maintain metabolism in dynamic artificial cell models.

An Exploratory Phase II Study of Eribulin Re-challenge After Short Term Therapy of 5-Fluorouracil for HER2 Negative, Advanced or Recurrent Breast Cancer.

BACKGROUND/AIM: In our previous study, first-line eribulin (ERI) showed 25 weeks of progression-free survival (PFS). This study investigated the efficacy and safety of ERI re-administration in metastatic breast cancer (MBC) patients. PATIENTS AND METHODS: HER2-negative MBC patients who had never received chemotherapy for MBC received first-line ERI for 18 weeks if they did not have disease progression, and then one cycle of S-1 before ERI re-administration. RESULTS: Twelve patients received ERI re-administration. The PFS of re-administered ERI was 13 weeks. Total duration of ERI use was 30 weeks. The incidence and severity of adverse events were consistent with previous reports. CONCLUSION: In the first-line setting, the total PFS of eribulin was extended by S-1 administration before disease progression, compared with that of our previous report.

Cellular compartment model for exploring the effect of the lipidic membrane on the kinetics of encapsulated biochemical reactions.

One of the important characteristics of the cellular system is that interactions between the plasma membrane and water-soluble molecules in the cytoplasm are enhanced by the confinement of the molecules to the small volume of the intracellular space. Studying this effect in a model cell system, we measured the time evolution of an enzymatic hydrolysis reaction and a cell-free protein synthesis reaction taking place in giant liposomes having various size and phospholipid compositions by a flow cytometry. This single vesicle-based assay of a large number of liposomes enabled us to examine the volume dependence of enclosed reactions in detail, revealing that the presence of specific lipid affected the specific kinetic parameters of encapsulated reactions.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

Neoadjuvant Chemotherapy With Nab-paclitaxel Plus Trastuzumab Followed by 5-Fluorouracil/Epirubicin/Cyclophosphamide for HER2-positive Operable Breast Cancer: A Multicenter Phase II Trial.

AIM: This study was conducted in order to evaluate the efficacy and safety of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) plus trastuzumab followed by 5-fluorouracil/ epirubicin/cyclophosphamide (FEC) in a neoadjuvant chemotherapy (NAC) setting for patients with human epidermal growth factor receptor 2 (HER2)-positive operable breast cancer. PATIENTS AND METHODS: Each patient received four cycles of 260 mg/m2 nab-paclitaxel with 6 mg/kg trastuzumab (8 mg/kg as the loading dose) every 3 weeks (q3w) followed by four cycles of FEC (500/100/500 mg/m2) q3w. The primary endpoint was pathological complete response (pCR) rate. RESULTS: Twenty-nine patients were analyzed for the efficacy and safety of this treatment. All patients completed four cycles of nab-paclitaxel and trastuzumab, and 28 patients completed four cycles of FEC. Twenty-seven patients subsequently underwent surgery. The pCR rate was 74.0%. The most frequent toxicity was sensory neuropathy (96.6%), but grade 3 neuropathy rate was 3.4%. CONCLUSION: Nab-paclitaxel plus trastuzumab followed by FEC in patients with HER2-positive operable breast cancer is considerably effective and well tolerated.

Replication of genetic information with self-encoded replicase in liposomes.

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qbeta replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in-liposome self-encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

Importance of translation-replication balance for efficient replication by the self-encoded replicase.

In all living systems, the genetic information is replicated by the self-encoded replicase (Rep); this can be said to be a self-encoding system. Recently, we constructed a self-encoding system in liposomes as an artificial cell model, consisting of a reconstituted translation system and an RNA encoding the catalytic subunit of Qbeta Rep and the RNA was replicated by the self-encoded Rep produced by the translation reaction. In this system, both the ribosome (Rib) and Rep bind to the same RNA for translation and replication, respectively. Thus, there could be a dilemma: effective RNA replication requires high levels of Rep translation, but excessive translation in turn inhibits replication. Herein, we actually observed the competition between the Rib and Rep, and evaluated the effect for RNA replication by constructing a kinetic model that quantitatively explained the behavior of the self-encoding system. Both the experimental and theoretical results consistently indicated that the balance between translation and replication is critical for an efficient self-encoded system, and we determined the optimum balance.

[A case of liver metastasis of rectal cancer demonstrating complete response for three years to 5-FU/l-leucovorin].

We report a case in which 5-fluorouracil/l-leucovorin (5-FU/l-LV) combination therapy was remarkably effective for non-resectable advanced rectal cancer with multiple liver metastasis. A 68-year-old man complaining of severe abdominal distension and abdominal pain was diagnosed as having ileus due to rectal cancer. We established a diagnosis of non-resectable rectal cancer with multiple liver metastasis and therefore performed only rectal colostomy. Systemic chemotherapy with 5-FU/l-LV was scheduled for a total of 22 courses postoperatively. After the chemotherapeutic regimen, a CT scan and colonofiberscopy revealed the primary lesions had disappeared, and a histological examination of biopsy confirmed that the patient had achieved complete response (CR).

Production of giant unilamellar vesicles by the water-in-oil emulsion-transfer method without high internal concentrations of sugars.

Giant unilamellar vesicles (GUVs) are large vesicles bounded by a single lipid bilayer, which have been used in various applications as artificial, cell-like compartments. The water-in-oil (w/o) emulsion-transfer method has been attracting attention as a method to prepare GUVs that can efficiently encapsulate macromolecules. For efficient GUV production by this method, non-physiological, high concentrations of sugars are usually required in the inner solution of the GUVs. These sugars limit the utility of the GUVs for a wide range of applications. In this study, we investigated various compositions of the inner and outer solutions to achieve efficient production without high concentrations of sugars through the w/o emulsion-transfer method. Firstly, we adjusted the osmotic pressure and density of the outer solution with NaCl and succeeded in increasing the proportion of GUVs and the absolute number in the prepared liposome population. Secondly, we increased the density of the inner solution with cytochrome c, but the proportion of GUVs and absolute number of vesicles did not increase. Thirdly, we increased the density of the inner and outer solutions with glycerol, which is membrane permeable and can be removed from GUVs, and succeeded in increasing the GUV proportion. These results provide useful information for the efficient preparation of GUVs that enclose a physiologically-relevant environment by the w/o emulsion-transfer method.