Correction: Degradation of YRA1 Pre-mRNA in the Cytoplasm Requires Translational Repression, Multiple Modular Intronic Elements, Edc3p, and Mex67p.

[This corrects the article DOI: 10.1371/journal.pbio.1000360.].

Direct Loading of iTEP-Delivered CTL Epitope onto MHC Class I Complexes on the Dendritic Cell Surface.

Cytotoxic T lymphocyte (CTL)-mediated immune responses are the primary defense mechanism against cancer and infection. CTL epitope peptides have been used as vaccines to boost CTL responses; however, the efficacy of these peptides is suboptimal. Under current vaccine formulation and delivery strategies, these vaccines are delivered into and processed inside antigen-presenting cells such as dendritic cells (DCs). However, the intracellular process is not efficient, which at least partially contributes to the suboptimal efficacy of the vaccines. Thus, we hypothesized that directly loading epitopes onto MHC class I complexes (MHC-Is) on the DC surface would significantly improve the efficacy of the epitopes because the direct loading bypasses inefficient intra-DC vaccine processing. To test the hypothesis, we designed an immune-tolerant elastin-like polypeptide (iTEP)-delivered CTL vaccine containing a metalloproteinase-9 (MMP-9)-sensitive peptide and an CTL epitope peptide. We found that the epitope was released from this MMP-sensitive vaccine through cleavage by DC-secreted MMP-9 outside of the DCs. The released epitopes were directly loaded onto MHC-Is on the DC surface. Ultimately, the MMP-sensitive vaccine strikingly increased epitope presentation by DCs by 7-fold and enhanced the epitope-specific CD8+ T-cell response by as high as 9.6-fold compared to the vaccine that was uncleavable by MMP. In summary, this novel direct-loading strategy drastically boosted vaccine efficacy. This study offered a new avenue to enhance CTL vaccines.

An Anti-Programmed Death-1 Antibody (αPD-1) Fusion Protein That Self-Assembles into a Multivalent and Functional αPD-1 Nanoparticle.

Cancer immune checkpoint therapy has achieved remarkable clinical successes in various cancers. However, current immune checkpoint inhibitors block the checkpoint of not only the immune cells that are important to cancer therapy but also the immune cells that are irrelevant to the therapy. Such an indiscriminate blockade limits the efficacy and causes the autoimmune toxicity of the therapy. It might be beneficial to use a carrier to target immune checkpoint inhibitors to cancer-reactive immune cells. Here, we explore a method to load the inhibitors into carriers. We used the anti-programmed death-1 antibody (αPD-1) as a model immune checkpoint inhibitor. First, we generated a recombinant single-chain variable fragment (scFv) of αPD-1. Then, we designed and generated a fusion protein consisting of the scFv and an amphiphilic immune-tolerant elastin-like polypeptide (iTEP). Because of the amphiphilic iTEP, the fusion was able to self-assemble into a nanoparticle (NP). The NP was proved to block the PD-1 immune checkpoint in vitro and in vivo. Particularly, the NP exacerbated diabetes development in nonobese diabetic mice as effectively as natural, intact αPD-1. In summary, we successfully expressed αPD-1 as a recombinant protein and linked αPD-1 to a NP, which lays a foundation to develop a delivery system to target αPD-1 to a subpopulation of immune cells.

Engineering of a self-adjuvanted iTEP-delivered CTL vaccine.

Cytotoxic T lymphocyte (CTL) epitope peptide-based vaccines are widely used in cancer and infectious disease therapy. We previously generated an immune-tolerant elastin-like polypeptides (iTEPs)-based carrier to deliver a peptide CTL vaccine and enhance the efficiency of the vaccine. To further optimize the vaccine carrier, we intended to potentiate its function by designing an iTEP-based carrier that was able to deliver adjuvant and a vaccine epitope as one molecule. Thus, we fused a 9-mer H100, a peptide derived from the high-mobility group box 1 protein (HMGB1) that could induce activation of dendritic cells (DCs), with an iTEP polymer to generate a new iTEP polymer named H100-iTEP. The H100-iTEP still kept the feature of reversible phase transition of iTEPs and should be able to be used as a polymer carrier to deliver peptide vaccines. The expression levels of CD80/CD86 on DCs were assessed using flow cytometry. The iTEP fusion-stimulated IL-6 secretion by DCs was measured with ELISA. Activation of antigen-specific CD8+ T cells induced by iTEP fusions was examined through a B3Z hybridoma cell activation assay. In vivo CTL activation promoted by iTEP fusions was detected by an IFN-γ-based ELISPOT assay. The iTEP fused with H100 could induce maturation of DCs in vitro as evidenced by increased CD80 and CD86 expression. The iTEP fusion also promoted activation of DCs by increasing secretion of a proinflammatory cytokine IL-6. The N-terminus or C-terminus fusion of H100 to iTEP had a similar effect and a reduced form of cysteine in iTEP fusions was required for DC stimulation. iTEP fusions potentiated a co-administrated CTL vaccine by increasing an antigen-specific CTL response in vitro and in vivo. When the H100-iTEP was fused to a CTL epitope to generate a one-molecule vaccine, this self-adjuvanted vaccine elicited a stronger antigen-specific CTL response than a vaccine adjuvanted by Incomplete Freund's Adjuvant. Thus, we have successfully generated a functional, one-molecule iTEP-based self-adjuvanted vaccine.

Treatment of type 1 myotonic dystrophy by engineering site-specific RNA endonucleases that target (CUG)(n) repeats.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of (CTG)n in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, which is transcribed as (CUG)n repeats that accumulate in the nucleus. The RNA repeats specifically sequester or change the expression levels of several RNA-binding proteins, leading to aberrant splicing of many target genes. In this study, we developed artificial site-specific RNA endonucleases (ASREs) that specifically bind and cleave (CUG)n repeats RNA. We have generated one ASRE that can target the expanded RNA repeats in DM1 patient cells and specifically degrade the pathogenic DMPK messenger RNAs with minimal effect on wild-type alleles. Such ASRE treatment significantly decreased the number of nuclear foci in DM1 patient cells and can reverse the missplicing of many genes affected in DM1 patients. Taken together, the application of ASRE provides a new route of gene therapy for DM1 treatment.

iTEP nanoparticle-delivered salinomycin displays an enhanced toxicity to cancer stem cells in orthotopic breast tumors.

Salinomycin (Sali) has selective toxicity to cancer stem cells (CSCs), a subpopulation of cancer cells that have been recently linked with tumor multidrug resistance (MDR). To utilize its selective toxicity for cancer therapy, we sought to devise a nanoparticle (NP) carrier to deliver Sali to solid tumors through the enhanced permeability and retention effect and, hence, to increase its exposure to CSCs. First, hydrophobic Sali was conjugated to a hydrophilic, immune-tolerant, elastin-like polypeptide (iTEP); the amphiphilic iTEP-Sali conjugates self-assemble into NPs. Next, free Sali was encapsulated into the NPs alone or with two additives, N,N-dimethylhexylamine (DMHA) and α-tocopherol. The coencapsulation significantly improved the loading efficiency and release profile of Sali. The resulting NPs of the coencapsulation, termed as iTEP-Sali NP3s, have an in vitro release half-life of 4.1 h, four times longer than iTEP-Sali NP2s, the NPs that have encapsulated Sali only. Further, the NP3 formulation increases the plasma area under curve and the tumor accumulation of Sali by 10 and 2.4 times, respectively. Lastly, these improved pharmacokinetic and tumor accumulation profiles are consistent with a boost of CSC-elimination effect of Sali in vivo. In NP3-treated 4T1 orthotopic tumors, the mean CSC frequency is 55.62%, a significant reduction from the mean frequencies of untreated tumors, 75.00%, or free Sali-treated tumors, 64.32%. The CSC-elimination effect of the NP3 can further translate to a delay of tumor growth. Given the role of CSCs in driving tumor MDR and recurrence, it could be a promising strategy to add the NP3 to conventional cancer chemotherapies to prevent or reverse the MDR.

Degradation of YRA1 Pre-mRNA in the cytoplasm requires translational repression, multiple modular intronic elements, Edc3p, and Mex67p.

Intron-containing pre-mRNAs are normally retained and processed in the nucleus but are sometimes exported to the cytoplasm and degraded by the nonsense-mediated mRNA decay (NMD) pathway as a consequence of their inclusion of intronic in-frame termination codons. When shunted to the cytoplasm by autoregulated nuclear export, the intron-containing yeast YRA1 pre-mRNA evades NMD and is targeted by a cytoplasmic decay pathway mediated by the decapping activator Edc3p. Here, we have elucidated this transcript-specific decay mechanism, showing that Edc3p-mediated YRA1 pre-mRNA degradation occurs independently of translation and is controlled through five structurally distinct but functionally interdependent modular elements in the YRA1 intron. Two of these elements target the pre-mRNA as an Edc3p substrate and the other three mediate transcript-specific translational repression. Translational repression of YRA1 pre-mRNA also requires the heterodimeric Mex67p/Mtr2p general mRNA export receptor, but not Edc3p, and serves to enhance Edc3p substrate specificity by inhibiting the susceptibility of this pre-mRNA to NMD. Collectively, our data indicate that YRA1 pre-mRNA degradation is a highly regulated process that proceeds through translational repression, substrate recognition by Edc3p, recruitment of the Dcp1p/Dcp2p decapping enzyme, and activation of decapping.

Diphtheria toxin-derived, anti-PD-1 immunotoxin, a potent and practical tool to selectively deplete PD-1+ cells.

Programmed death-1 (PD-1), an immune checkpoint receptor, is expressed on activated lymphocytes, macrophages, and some types of tumor cells. While PD-1+ cells have been implicated in outcomes of cancer immunity, autoimmunity, and chronic infections, the exact roles of these cells in various physiological and pathological processes remain elusive. Molecules that target and deplete PD-1+ cells would be instrumental in defining the roles unambiguously. Previously, an immunotoxin has been generated for the depletion of PD-1+ cells though its usage is impeded by its low production yield. Thus, a more practical molecular tool is desired to deplete PD-1+ cells and to examine functions of these cells. We designed and generated a novel anti-PD1 diphtheria immunotoxin, termed PD-1 DIT, targeting PD-1+ cells. PD-1 DIT is comprised of two single chain variable fragments (scFv) derived from an anti-PD-1 antibody, coupled with the catalytic and translocation domains of the diphtheria toxin. PD-1 DIT was produced using a yeast expression system that has been engineered to efficiently produce protein toxins. The yield of PD-1 DIT reached 1-2 mg/L culture, which is 10 times higher than the previously reported immunotoxin. Flow cytometry and confocal microscopy analyses confirmed that PD-1 DIT specifically binds to and enters PD-1+ cells. The binding avidities between PD-1 DIT and two PD-1+ cell lines are approximately 25 nM. Moreover, PD-1 DIT demonstrated potent cytotoxicity toward PD-1+ cells, with a half maximal effective concentration (EC50 ) value of 1 nM. In vivo experiments further showed that PD-1 DIT effectively depleted PD-1+ cells and enabled mice inoculated with PD-1+ tumor cells to survive throughout the study. Our findings using PD-1 DIT revealed the critical role of pancreatic PD-1+ T cells in the development of type-1 diabetes (T1D). Additionally, we observed that PD-1 DIT treatment ameliorated relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), a mouse model of relapsing-remitting multiple sclerosis (RR-MS). Lastly, we did not observe significant hepatotoxicity in mice treated with PD-1 DIT, which had been reported for other immunotoxins derived from the diphtheria toxin. With its remarkable selective and potent cytotoxicity toward PD-1+ cells, coupled with its high production yield, PD-1 DIT emerges as a powerful biotechnological tool for elucidating the physiological roles of PD-1+ cells. Furthermore, the potential of PD-1 DIT to be developed into a novel therapeutic agent becomes evident.

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains.

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.

Antibody-mediated depletion of programmed death 1-positive (PD-1+) cells.

PD-1 immune checkpoint has been intensively investigated in pathogenesis and treatments for cancer and autoimmune diseases. Cells that express PD-1 (PD-1+ cells) draw ever-increasing attention in cancer and autoimmune disease research although the role of PD-1+ cells in the progression and treatments of these diseases remains largely ambiguous. One definite approach to elucidate their roles is to deplete these cells in disease settings and examine how the depletion impacts disease progression and treatments. To execute the depletion, we designed and generated the first depleting antibody (D-αPD-1) that specifically ablates PD-1+ cells. D-αPD-1 has the same variable domains as an anti-mouse PD-1 blocking antibody (RMP1-14). The constant domains of D-αPD-1 were derived from mouse IgG2a heavy and κ-light chain, respectively. D-αPD-1 was verified to bind with mouse PD-1 as well as mouse FcγRIV, an immuno-activating Fc receptor. The cell depletion effect of D-αPD-1 was confirmed in vivo using a PD-1+ cell transferring model. Since transferred PD-1+ cells, EL4 cells, are tumorigenic and EL4 tumors are lethal to host mice, the depleting effect of D-αPD-1 was also manifested by an absolute survival among the antibody-treated mice while groups receiving control treatments had median survival time of merely approximately 30 days. Furthermore, we found that D-αPD-1 leads to elimination of PD-1+ cells through antibody-dependent cell-mediate phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) mechanisms. These results, altogether, confirmed the specificity and effectiveness of D-αPD-1. The results also highlighted that D-αPD-1 is a robust tool to study PD-1+ cells in cancer and autoimmune diseases and a potential therapeutic for these diseases.

Promotion of CTL epitope presentation by a nanoparticle with environment-responsive stability and phagolysosomal escape capacity.

Vaccines that induce cytotoxic T lymphocyte (CTL)-mediated immune responses constitute an important class of medical tools to fend off diseases like infections and malignancy. Epitope peptides, as a format of CTL vaccines, are being tested preclinically and clinically. To elicit CTL responses, epitope vaccines go through an epitope presentation pathway in dendritic cells (DCs) that has multiple bottleneck steps and hence is inefficient. Here, we report the development of a strategy to overcome one of these barriers, phagolysosomal escape in DCs. First, we furnished a previously established carrier-an immune-tolerant elastin-like polypeptide nanoparticle (iTEP NP)-with the peptides that are derived from the DNA polymerase of herpes simplex virus 1 (Pol peptides). Pol peptides were reported to facilitate phagolysosomal escape. In this study, while we found that Pol peptides promoted the CTL epitope presentation; we also discovered Pol peptides disrupted the formation of the iTEP NP. Thus, we engineered a series of new iTEPs and identified several iTEPs that could accommodate Pol peptides and maintain their NP structure at the same time. We next optimized one of these NPs so that its stability is responsive to its redox environment. This environment-responsive NP further strengthened the CTL epitope presentation and CTL responses. Lastly, we revealed how this NP and Pol peptides utilized biological cues of phagolysosomes to realize phagolysosomal escape and epitope release. In summary, we developed iTEP NP carriers with a new phagolysosomal escape function. These carriers, with their priorly incorporated functions, resolve three bottleneck issues in the CTL epitope presentation pathway: vaccine uptake, phagolysosomal escape, and epitope release.

Depletion of PD-1-positive cells ameliorates autoimmune disease.

Targeted suppression of autoimmune diseases without collateral suppression of normal immunity remains an elusive yet clinically important goal. Targeted blockade of programmed-cell-death-protein-1 (PD-1)-an immune checkpoint factor expressed by activated T cells and B cells-is an efficacious therapy for potentiating immune activation against tumours. Here we show that an immunotoxin consisting of an anti-PD-1 single-chain variable fragment, an albumin-binding domain and Pseudomonas exotoxin targeting PD-1-expressing cells, selectively recognizes and induces the killing of the cells. Administration of the immunotoxin to mouse models of autoimmune diabetes delays disease onset, and its administration in mice paralysed by experimental autoimmune encephalomyelitis ameliorates symptoms. In all mouse models, the immunotoxin reduced the numbers of PD-1-expressing cells, of total T cells and of cells of an autoreactive T-cell clone found in inflamed organs, while maintaining active adaptive immunity, as evidenced by full-strength immune responses to vaccinations. The targeted depletion of PD-1-expressing cells contingent to the preservation of adaptive immunity might be effective in the treatment of a wide range of autoimmune diseases.

Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo.

Dendritic cell (DC)-based cytotoxic T lymphocyte (CTL) epitope vaccines are effective to induce CTL responses but require complex ex vivo DC preparation and epitope-loading. To take advantage of DC-based epitope vaccines without involving the ex vivo procedures, we aimed to develop carriers to directly load CTL epitopes onto DCs in vivo. Here, we first engineered a carrier consisting of a hydrophilic polypeptide, immune-tolerant elastin-like polypeptide (iTEP) and a substrate peptide of matrix metalloproteinases-9 (sMMP). The iTEP was able to solubilize CTL epitopes. CTL epitopes were connected to the carrier, iTEP-sMMP, through sMMP so that the epitopes can be cleaved from the carrier by MMP-9. iTEP-sMMP was found to release its epitope payloads in the DC culture media, which contained MMP-9 released from DCs. iTEP-sMMP allowed for the direct loading of CTL epitopes onto the surface MHC class I complexes of DCs. Importantly, iTEP-sMMP resulted in greater epitope presentation by DCs both in vitro and in vivo than a control carrier that cannot directly load epitopes. iTEP-sMMP also induced 2-fold stronger immune responses than the control carrier. To further enhance the direct epitope-loading strategy, we furnished iTEP-sMMP with an albumin-binding domain (ABD) and found the new carrier, ABD-iTEP-sMMP, had greater lymph node (LN) accumulation than iTEP-sMMP. ABD-iTEP-sMMP also resulted in greater immune responses than iTEP-sMMP by 1.5-fold. Importantly, ABD-iTEP-sMMP-delivered CTL epitope vaccine induced stronger immune responses than free CTL epitope vaccine. Taken together, these carriers utilized two physiological features of DCs to realize direct epitope-loading in vivo: the accumulation of DCs in LNs and MMP-9 released from DCs. These carriers are a potential substitute for DC-based CTL epitope vaccines.

An Albumin-binding Polypeptide Both Targets Cytotoxic T Lymphocyte Vaccines to Lymph Nodes and Boosts Vaccine Presentation by Dendritic Cells.

Rationale: Albumin-binding carriers have been shown to target cytotoxic T lymphocyte (CTL) vaccines to lymph nodes (LNs) and improve the efficacy of the vaccines. However, it was not clear whether the improved efficacy is solely due to the LN targeting, which prompted this study. Methods: First, we generated a fusion protein consisting of an albumin-binding domain (ABD) and an immune-tolerant elastin-like polypeptide (iTEP). Then, we examined the binding between this fusion protein, termed ABD-iTEP, and mouse serum albumin (MSA). Next, we evaluated the accumulation of ABD-iTEP in LNs and dendritic cells (DCs) in the LNs. We also analyzed antigen presentation and in vitro T cell activation of vaccines that were delivered by ABD-iTEP and investigated possible underlying mechanisms of the presentation and activation results. Last, we measured CTL responses induced by ABD-iTEP-delivered vaccines in vivo. Results: ABD-iTEP bound with MSA strongly with an affinity of 1.41 nM. This albumin-binding carrier, ABD-iTEP, accumulated in LNs 3-fold more than iTEP, a control carrier that did not bind with albumin. ABD-iTEP also resulted in 4-fold more accumulation in DCs in the LNs than iTEP. Most importantly, ABD-iTEP drastically enhanced the antigen presentation of its vaccine payloads and the T cell activation induced by its payloads. The enhancement was dependent on the formation of the complex between MSA and ABD-iTEP. Meanwhile, the MSA/ABD-iTEP complex was found to have increased stability in acidic subcellular compartments and increased cytosolic accumulation in DCs, which might explain the enhanced vaccine presentation resulting from the complex. Finally, when ABD-iTEP was used to deliver CTL vaccines derived from both self- and non-self-antigens, it boosted the vaccine-induced responses by 2-fold in either case. Conclusion: ABD-iTEP not only targets vaccines to LNs but also promotes the presentation of the vaccines by DCs. Albumin-binding carriers have more than one mechanism to boost the efficacy of CTL vaccines.

An iTEP-salinomycin nanoparticle that specifically and effectively inhibits metastases of 4T1 orthotopic breast tumors.

Cancer stem cell (CSC) inhibitors are a new category of investigational drugs to treat metastasis. Salinomycin (Sali) is one of most studied CSC inhibitors and has reached clinical tests. Several drug carriers have been developed to improve efficacy of Sali. However, Sali has not been shown to inhibit metastasis from orthotopic tumors, the gold standard for metastasis. To fill this gap, we developed an immune-tolerant, elastin-like polypeptide (iTEP)-based nanoparticle (iTEP-Sali-ABA NP) that released 4-(aminomethyl)benzaldehyde-modified Sali (Sali-ABA) under acidic conditions. We found that the NP increased the area under the curve (AUC) of Sali-ABA by 30-fold and the tumor accumulation by 3.4-fold. Furthermore, no metastasis was detected in any of the mice given the NP. However, all the mice died of primary tumor burdens. To overcome primary tumor growth and improve the overall survival, we applied a combination therapy consisting of the iTEP-Sali-ABA NP and iTEP NP-delivered paclitaxel. This therapy effectively retarded primary tumor growth, and most importantly, improved the overall survival. In conclusion, delivery of Sali-ABA by the NP, alone or in combination with paclitaxel, was more effective than free Sali-ABA in decreasing metastasis and increasing survival. This iTEP-Sali-ABA NP represents a novel and clinically promising therapy to combat metastasis.

A Comparison Study of iTEP Nanoparticle-Based CTL Vaccine Carriers Revealed a Surprise Relationship between the Stability and Efficiency of the Carriers.

Vaccine carriers have been shown to enhance cytotoxic T lymphocyte (CTL) epitope peptide vaccines by addressing intrinsic limitations of the vaccines. We have previously developed an immune-tolerant elastin-like polypeptide (iTEP)-based nanoparticle (NP) as an effective and unique CTL vaccine carrier. The NP is unique for its humoral immune tolerance, flexible structure, and ability to deliver CTL vaccines as polypeptide fusions. Here, we aimed to improve the NP by increasing its stability since we found it was not stable. We thus generated a more stable iTEP NP (ST-NP) and used it to deliver a CTL peptide vaccine, SIINFEKL. However, we surprisingly found that the ST-NP had a lower efficiency than the previously developed, marginally stable iTEP NP (MS-NP) in terms of promoting vaccine presentation and vaccine-induced CTL responses. On the other hand, dendritic cells (DCs) showed preferential uptake of the ST-NP but not the MS-NP. To develop an iTEP vaccine carrier that outperforms both the MS-NP and the ST-NP, we devised an iTEP NP that has a changeable stability responsive to a cytosolic, reductive environment, termed reductive environment-dependent NP or RED-NP. The RED-NP showed an intermediate ability to promote vaccine presentation and T cell responses in vitro between the MS-NP and the ST-NP. However, the RED-NP induced the strongest CTL responses in vivo among all three NPs. In conclusion, iTEP NPs that have a dynamically changeable stability are most effective to deliver and enhance CTL peptide vaccines. The work also demonstrated the versatile nature of iTEP vaccine carriers.

[Clinical efficacy of shenzhe zhuyun mixture in treating esophageal lesion in patients with systemic sclerosis].

OBJECTIVE: To investigate the clinical efficacy of Shenzhe Zhuyun mixture (SZM) in treating esophageal lesion in patients with systemic sclerosis. METHODS: Sixty-four patients conforming to the inclusion criteria were randomly divided into 2 groups, the treated group and the control group treated respectively by SZM and cisapride, with the blank preparation imitating the contrast one, the treatment course to both groups was 2 months. The clinical efficacy, the changes in symptom scores of esophageal lesion, the orthostatic and clinostatic time of barium passing through esophagus, the clinostatic esophageal emptying index of barium, and the widest transverse diameter of ectatic esophageal segment, as well as the influence of treatment on patients' quality of life were observed. RESULTS: The total effective rate in the treated group was superior to that in the control group (96.9% vs 56.2%, P < 0.01). Symptom of esophageal lesion was significantly improved after treatment in both groups (P<0.01), and the improvement in the treated group was better than that in the control group (P <0.01). The time of Barium passing through esophagus, both orthostatic and clinostatic, and the esophageal emptying index of barium were improved in the treated group (P < 0.01), but the change of the widest transverse diameter of ectatic esophagus was insignificantly improved. While in the control group, excepting the clinostatic time of barium passing through was improved (P<0.01), the other indexes showed no obvious change. Patients' quality of life was significantly improved in the treated group, but improvement didn't revealed in the control group. CONCLUSION: SZM can reinforce esophageal dynamic function in patients with systemic sclerosis, it can be an effective TCM prescription in treating esophageal lesion for them.

[The effect of nitric oxide on acute lung injury following ischemia/reperfusion of hind limbs in the rat].

On a model of reperfusion after ischemia in the hind limbs (LIR) of rats, we used aminoguanidine (AG) which inhibits nitric oxide synthase (NOS) and L-arginine (L-Arg), one of the substrates in the process of nitric oxide synthesis, to observe the changes in NO, NOS, malondialdehyde (MDA), myeloperoxidase (MPO) and wet/dry ratio (W/D) in both skeletal muscles and the lung as well as the changes in phosphatidyl choline (PC) of lung surfactant. The morphologic changes were observed with microscopy. It was observed that the values of NOS, MPO, MDA of the muscle and lung in LIR group increased significantly and the content of PC decreased obviously compared with those of the normal control. Pulmonary observation revealed that after LIR leucocyte assembling and infiltration took place, which was dominated by polymorphocytes with broadened pulmonary interstitial tissue. In LIR+L-Arg group the above changes were reversed, and in LIR+AG group the injuries became more serious. The results obtained suggest that the activity of NOS and the production of NO following ischemia/reperfusion of hind limbs increased significantly, and that the endogenous NO may play a protective role during the early stage of acute lung injury after LIR.

Generation of designer receptors exclusively activated by designer drugs (DREADDs) using directed molecular evolution.

G protein-coupled receptors (GPCRs) and their signal transductions are important for both physiological and pathological processes in neuron systems. Neuronal GPCRs activated by synthetic ligands have been created by designed mutagenesis for studying their functions and signal pathways. However, these engineered GPCRs have problems, such as their high constitutive activity. To overcome this drawback, a new generation of receptors termed designer receptors exclusively activated by designer drugs (DREADDs), have been designed. DREADDs are exclusively activated by synthetic ligands, but are insensitive to their endogenous ligand and have no constitutive activity, which provides the ability to selectively modulate signal transduction of certain GPCRs in vitro and in vivo. This protocol provides detailed instructions for creating DREADDs using directed molecular evolution. The procedures to generate DREADDS include GPCR functional expression in yeast, mutant GPCR library generation, and high-throughput yeast screening. These methods are general and suitable for any GPCRs that can be functionally expressed in yeast.

Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.