Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit.

The -1 programmed ribosomal frameshifting (-1 PRF) has been explored as a gene regulatory circuit for synthetic biology applications. The -1 PRF usually uses an RNA pseudoknot structure as the frameshifting stimulator. Finding a ligand-responsive pseudoknot with efficient -1 PRF activity is time consuming and is becoming a bottleneck for its development. Inserting a guanine to guanine (GG)-mismatch pair in the 5'-stem of a small frameshifting pseudoknot could attenuate -1 PRF activity by reducing stem stability. Thus, a ligand-responsive frameshifting pseudoknot can be built using GG-mismatch-targeting small molecules to restore stem stability. Here, a pseudoknot requiring stem-loop tertiary interactions for potent frameshifting activity was used as the engineering template. This considerably amplified the effect of mismatch destabilization, and led to creation of a mammalian -1 PRF riboswitch module capable of mediating premature translation termination as a synthetic regulatory mode. Application of the synthetic circuit allowed ligand-dependent ATF6N mimic formation for the activation of protein folding-related genes involved in the unfolded protein response without an ER-stress inducing agent. With the availability of mismatch-targeting molecules, the tailored module thus paves the way for various mismatch plug-ins to streamline highly efficient orthogonal ligand-dependent -1 PRF stimulator development in the synthetic biology toolbox.

Adjuvant whole breast radiotherapy with simultaneous integrated boost to tumor bed with intensity modulated radiotherapy technique in elderly breast cancer patients.

BACKGROUND: Adjuvant whole breast radiotherapy is the standard of care for breast cancer patients after partial mastectomy. Intensity-modulated radiation therapy (IMRT) has been reported to reduce acute toxicities compared to conventional radiotherapy. IMRT with simultaneous integrated boost (SIB) technique can deliver higher doses to tumor bed and irradiate whole breast with a lower dose level to shorten overall treatment duration. This study presents the long-term results of adjuvant IMRT with SIB in elderly breast cancer patients who received partial mastectomy. METHODS: From January 2007 to January 2018, 93 elder breast cancer patients (≥65-year-old) who received IMRT with SIB technique after partial mastectomy were reviewed retrospectively. The axillary areas were managed with either sentinel lymph node biopsies or axillary lymph node dissection. The dose to whole breast was 50.4 Gy in 28 fractions in all patients and the dose to tumor bed was 61.6 to 66.4 Gy in 28 fractions. The primary end point is locoregional control. Secondary end points include: overall survival, breast cancer-specific survival, distant-metastases-free survival, disease-free survival, and acute and chronic toxicities. RESULTS: The median follow-up was 56.1 months. One patient had ipsilateral breast tumor recurrence, 3 patients had regional lymph node recurrence, and 9 patients had distant metastases. Death occurred in 5 patients, including 3 patients died of breast cancer progression. Five-year overall survival is 96.3% and 5-year locoregional recurrence-free survival is 96.4%. The 5-year breast cancer specific survival and 5-year distant metastases-free survival is 97.5% and 87.2%, respectively. Seven patients developed second primary cancer after RT. Eighty-one point seven percent patients had acute grade 1 dermatitis while 18.3% suffered from grade 2 dermatitis. The incidence of grade 1 pneumonitis and grade 1 stomatitis was 4.3% and 8.6%, respectively. CONCLUSIONS: Adjuvant IMRT with SIB technique is a safe and effective treatment strategy for elderly breast cancer patients after partial mastectomy.

A topology-based method to mitigate the dosimetric uncertainty caused by the positional variation of the boost volume in breast conservative radiotherapy.

BACKGROUND: To improve local control rate in patients with breast cancer receiving adjuvant radiotherapy after breast conservative surgery, additional boost dose to the tumor bed could be delivered simultaneously via the simultaneous integrated boost (SIB) modulated technique. However, the position of tumor bed kept changing during the treatment course as the treatment position was aligned to bony anatomy. This study aimed to analyze the positional uncertainties between bony anatomy and tumor bed, and a topology-based approach was derived to stratify patients with high variation in tumor bed localization. METHODS: Sixty patients with early-stage breast cancer or ductal carcinoma in situ were enrolled. All received adjuvant whole breast radiotherapy with or without local boost via SIB technique. The delineation of tumor bed was defined by incorporating the anatomy of seroma, adjacent surgical clips, and any architectural distortion on computed tomography simulation. A total of 1740 on-board images were retrospectively analyzed. Positional uncertainty of tumor bed was assessed by four components: namely systematic error (SE), and random error (RE), through anterior-posterior (AP), cranial-caudal (CC), left-right (LR) directions and couch rotation (CR). Age, tumor location, and body-mass factors including volume of breast, volume of tumor bed, breast thickness, and body mass index (BMI) were analyzed for their predictive role. The appropriate margin to accommodate the positional uncertainty of the boost volume was assessed, and the new plans with this margin for the tumor bed was designed as the high risk planning target volume (PTV-H) were created retrospectively to evaluate the impact on organs at risk. RESULTS: In univariate analysis, a larger breast thickness, larger breast volume, higher BMI, and different tumor locations correlated with a greater positional uncertainty of tumor bed. However, BMI was the only factor associated with displacements of surgical clips in the multivariate analysis and patients with higher BMI were stratified as high variation group. When image guidance was aligned to bony structures, the SE and RE of clip displacement were consistently larger in the high variation group. The corresponding PTV-H margins for the high- and low-variation groups were 7, 10, 10 mm and 4, 9, 6 mm in AP, CC, LR directions, respectively. The heart dose between the two plans was not significantly different, whereas the dosimetric parameters for the ipsilateral lung were generally higher in the new plans. CONCLUSIONS: In patients with breast cancer receiving adjuvant radiotherapy, a higher BMI is associated with a greater positional uncertainty of the boost tumor volume. More generous margin should be considered and it can be safely applied through proper design of beam arrangement with advanced treatment techniques.

Synergetic regulation of translational reading-frame switch by ligand-responsive RNAs in mammalian cells.

Distinct translational initiation mechanisms between prokaryotes and eukaryotes limit the exploitation of prokaryotic riboswitch repertoire for regulatory RNA circuit construction in mammalian application. Here, we explored programmed ribosomal frameshifting (PRF) as the regulatory gene expression platform for engineered ligand-responsive RNA devices in higher eukaryotes. Regulation was enabled by designed ligand-dependent conformational rearrangements of the two cis-acting RNA motifs of opposite activity in -1 PRF. Particularly, RNA elements responsive to trans-acting ligands can be tailored to modify co-translational RNA refolding dynamics of a hairpin upstream of frameshifting site to achieve reversible and adjustable -1 PRF attenuating activity. Combined with a ligand-responsive stimulator, synthetic RNA devices for synergetic translational-elongation control of gene expression can be constructed. Due to the similarity between co-transcriptional RNA hairpin folding and co-translational RNA hairpin refolding, the RNA-responsive ligand repertoire provided in prokaryotic systems thus becomes accessible to gene-regulatory circuit construction for synthetic biology application in mammalian cells.

The reduction in σ-promoter recognition flexibility as induced by core RNAP is required for σ to discern the optimal promoter spacing.

Sigma (σ) factors are bacterial transcription initiation factors that direct transcription at cognate promoters. The promoters recognized by primary σ are composed of -10 and -35 consensus elements separated by a spacer of 17±1 bp for optimal activity. However, how the optimal promoter spacing is sensed by the primary σ remains unclear. In the present study, we examined this issue using a transcriptionally active Bacillus subtilis N-terminally truncated σA (SND100-σA). The results of the present study demonstrate that SND100-σA binds specifically to both the -10 and -35 elements of the trnS spacing variants, of which the spacer lengths range from 14 to 21 bp, indicating that simultaneous and specific recognition of promoter -10 and -35 elements is insufficient for primary σ to discern the optimal promoter spacing. Moreover, shortening in length of the flexible linker between the two promoter DNA-binding domains of σA also does not enable SND100-σA to sense the optimal promoter spacing. Efficient recognition of optimal promoter spacing by SND100-σA requires core RNAP (RNA polymerase) which reduces the flexibility of simultaneous and specific binding of SND100-σA to both promoter -10 and -35 elements. Thus the discrimination of optimal promoter spacing by σ is core-dependent.

The stable association of virion with the triple-gene-block protein 3-based complex of Bamboo mosaic virus.

The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.

Regulation of programmed ribosomal frameshifting by co-translational refolding RNA hairpins.

RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3' direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) -1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate-1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for-1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing-1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study's findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.

The core-independent promoter-specific interaction of primary sigma factor.

Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. Most σ functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, σ(A), by itself, is able to interact specifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the σ(A) is -10 specific. However, the promoter -10 specific interaction is unable to allow the σ(A) to discern the optimal promoter spacing. To fulfill this goal, the σ(A) requires assistance from core RNA polymerase (RNAP). The ability of σ, by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function.

The two conserved cysteine residues of the triple gene block protein 2 are critical for both cell-to-cell and systemic movement of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which is known to be required for the cell-to-cell movement of potexviruses. This protein has two conserved Cys residues, Cys-109 and Cys-112, at its C-terminal tail, which is supposed to be exposed on the outer surface of the endoplasmic reticulum (ER) membrane and ER-derived granular vesicles. In this study, we investigated the importance of these two Cys residues on the cell-to-cell and systemic movement of BaMV. Our results indicate that the Cys-to-Ala substitutions in TGBp2 make the cell-to-cell movement of BaMV relatively inefficient and the systemic movement of BaMV severely inhibited. Moreover, the defect in systemic movement is attributed to the inefficient transport of viral RNA in the phloem of petiole. Clearly, TGBp2 is critical not only for the cell-to-cell but also for the systemic movement of BaMV. In addition, the conserved Cys residues are important for the functioning of TGBp2.

Characterization of the RNA-binding properties of the triple-gene-block protein 2 of Bamboo mosaic virus.

The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction.

Topological properties of the triple gene block protein 2 of Bamboo mosaic virus.

The triple gene block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) has been proposed to be a transmembrane protein; however, its features remain unclear. Here, we used biochemical approaches to determine its topological properties. Our data reveal that TGBp2 is mainly associated with the endoplasmic reticulum membrane. The resistance of TGBp2 in proteoliposomes, prepared from both the BaMV-infected tissues and in vitro reconstitution system, to both chemical extraction and trypsin digestion confirmed that it is indeed an integral membrane protein. On the basis of the minor change in the size of the major stable TGBp2-derived tryptic fragment from the monomeric TGBp2, as well as the sensitivity of the cysteine residues at the C-terminal tail of TGBp2 to maleimide modification, we suggest that TGBp2 adopts a topology with both its short N- and C-terminal tails exposed to the outer surface of the endoplasmic reticulum. Moreover, TGBp2 is able to self-assemble as revealed by the significant increase in multimeric TGBp2 when the TGBp2-containing proteoliposomes were treated with chemical crosslinker or oxidation agent.