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Objective: To compare the safety and effectiveness of robot‐assisted minimally invasive transforaminal lumbar interbody fusion (Mis‐TLIF) and oblique lumbar interbody fusion (OLIF) for the treatment of single‐level lumbar degenerative spondylolisthesis (LDS). Methods: This is a retrospective study. Between April 2018 and April 2020, a total of 61 patients with single‐level lumbar degenerative spondylolisthesis and treated with robot‐assisted OLIF (28 cases, 16 females, 12 males, mean age 50.4 years) or robot‐assisted Mis‐TLIF (33 cases, 18 females, 15 males, mean age 53.6 years) were enrolled and evaluated. All the pedicle screws were implanted percutaneously assisted by the TiRobot system. Surgical data included the operation time, blood loss, and length of postoperative hospital stay. The clinical and functional outcomes included Oswestry Disability Index (ODI), Visual Analog scores (VAS) for back and leg pain, complication, and patient's satisfaction. Radiographic outcomes include pedicle screw accuracy, fusion status, and disc height. These data were collected before surgery, at 1 week, 3 months, 6 months, and 12 months postoperatively. Results: There were no significantly different results in preoperative measurement between the two groups. There was significantly less blood loss (142.4 ± 89.4 vs 291.5 ± 72.3 mL, P < 0.01), shorter hospital stays (3.2 ± 1.8 vs 4.2 ± 2.5 days, P < 0.01), and longer operative time (164.9 ± 56.0 vs 121.5 ± 48.2 min, P < 0.01) in OLIF group compared with Mis‐TLIF group. The postoperative VAS scores and ODI scores in both groups were significantly improved compared with preoperative data (P < 0.05). VAS scores for back pain were significantly lower in OLIF group than Mis‐TLIF group at 1 week (2.8 ± 1.2 vs 3.5 ± 1.6, P < 0.05) and 3 months postoperatively (1.6 ± 1.0 vs 2.1 ± 1.1, P < 0.05), but there was no significant difference at further follow‐ups. ODI score was also significantly lower in OLIF group than Mis‐TLIF group at 3 months postoperatively (22.3 ± 10.0 vs 26.1 ± 12.8, P < 0.05). There was no significant difference in the proportion of clinically acceptable screws between the two groups (97.3% vs 96.2%, P = 0.90). At 1 year, the OLIF group had a higher interbody fusion rate compared with Mis‐TLIF group (96.0% vs 87%, P < 0.01). Disc height was significantly higher in the OLIF group than Mis‐TLIF group (12.4 ± 3.2 vs 11.2 ± 1.3 mm, P < 0.01). Satisfaction rates at 1 year exceeded 90% in both groups and there was no significant difference (92.6% for OLIF vs 91.2% for Mis‐TLIF, P = 0.263). Conclusion: Robot‐assisted OLIF and Mis‐TLIF both have similar good clinical outcomes, but OLIF has the additional benefits of less blood loss, less postoperative hospital stays, higher disc height, and higher fusion rates. Robots are an effective tool for minimally invasive spine surgery. [ABSTRACT FROM AUTHOR]

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4–7 nt) while marginally increasing long abortives (9–11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP Kd by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1–2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation. [ABSTRACT FROM AUTHOR]

Methylene blue (MB) is a tricyclic heteroaromatic photosensitizer with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to salmon sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the K value of 5.6 × 10 M. The photobleaching processes of MB and its DNA complex have also been studied, which indicate that the photobleaching of MB and its DNA complex proceed with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB. [ABSTRACT FROM AUTHOR]

Abstract: Understanding the mechanism and fidelity of transcription by the RNA polymerase (RNAP) requires measurement of the dissociation constant (K d) of correct and incorrect NTPs and their incorporation rate constants (k pol). Currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. Here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. The fluorescent adenine analogue 2-aminopurine was incorporated at various single positions in the template or the nontemplate strand of the promoter-free elongation substrate. On addition of the correct NTP to the T7 RNAP-DNA, 2-aminopurine fluorescence increased rapidly and exponentially with a rate constant similar to the RNA extension rate obtained from the radiometric assay. The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. Using this assay, we report the k pol and K d of all four correct NTP additions by T7 RNAP, which showed a range of values of 145–190 s−1 and 28–124 μM, respectively. The fluorescent elongation substrates were used to determine the misincorporation kinetics as well, which showed that T7 RNAP discriminates against incorrect NTP both at the nucleotide binding and incorporation steps. The fluorescence-based assay should be generally applicable to all DNA-dependent RNAPs, as they use similar elongation substrates. It can be used to elucidate the mechanism, fidelity, and sequence dependency of transcription and is a rapid means to screen for inhibitors of RNAPs for therapeutic purposes. [ABSTRACT FROM AUTHOR]

The mitochondrial RNA polymerase (mtRNAP) of Saccharomyces cerevisiae, consisting of a complex of Rpo41 and Mtf1, is homologous to the phage single polypeptide T7/T3 RNA polymerases. The yeast mtRNAP recognizes a conserved nonanucleotide sequence to initiate specific transcription. In this work, we have defined the region of the nonanucleotide that is melted by the mtRNAP using 2-aminopurine (2AP) fluorescence that is sensitive to changes in base stacking interactions. We show that mtRNAP spontaneously melts the promoter from -4 to +2 forming a bubble around the transcription start site at + 1. The location and size of the DNA bubble in this open complex of the mtRNAP closely resembles that of the T7 RNA polymerase. We show that DNA melting requires the simultaneous presence of Rpo41 and Mtf1. Adding the initiating nucleotide AlP does not expand the size of the initially melted DNA, but the initiating nucleotide differentially affects base stacking interactions at -1 and -2. Thus, the promoter structure upstream of the transcription start site is slightly rearranged during early initiation from its structure in the pre-initiation stage. Unlike on the duplex promoter, Rpo41 alone was able to form a competent open complex on a pre-melted promoter. The results indicate that Rpo41 contains the elements for recognizing the melted promoter through interactions with the template strand. We propose that Mtf1 plays a role in base pair disruption during the early stages of open complex formation. [ABSTRACT FROM AUTHOR]

Summary: Elucidating the mechanism of transcription initiation by RNA polymerases (RNAP) is essential for understanding gene transcription and regulation. Although several models, such as DNA scrunching, RNAP translation, and RNAP rotation, have been proposed, the mechanism of initiation by T7 RNAP has remained unclear. Using ensemble and single-molecule Förster resonance energy transfer (FRET) studies, we provide evidence for concerted DNA scrunching and rotation during initiation by T7 RNAP. A constant spatial distance between the upstream and downstream edges of initiation complexes making 4–7 nt RNA supports the DNA scrunching model, but not the RNAP translation or the pure rotation model. DNA scrunching is accompanied by moderate hinging motion (18° ± 4°) of the promoter toward the downstream DNA. The observed stepwise conformational changes provide a basis to understand abortive RNA synthesis during early stages of initiation and promoter escape during the later stages that allows transition to processive elongation. [Copyright &y& Elsevier]

Abstract: Bacteriophage T7 RNA polymerase undergoes major conformational changes as transcription proceeds from initiation to elongation. Using limited trypsin digestion and stopped-flow fluorescence kinetic methods, we have monitored promoter release, initial bubble collapse, and refolding of the 152–205 region (subdomain H), the latter being important for RNA channel formation. The kinetic studies show that the conformational changes are temporally coupled, commencing at the synthesis of 9 nt and completing by the synthesis of 12 nt of RNA. The temporal coupling of initial bubble collapse and RNA channel formation is proposed to facilitate proper binding of the RNA dissociated from the late initiation complexes into the RNA channel. Using promoter mutations, we have determined that promoter contacts are broken sequentially during transition from initiation to elongation. The specificity loop interactions are broken after synthesis of 8 nt or 9 nt of RNA, whereas the upstream promoter contacts persists up to synthesis of 12 nt of RNA. Both promoter contacts need to be broken for transition into elongation. The A-15C mutation resulted in efficient transition to elongation by synthesis of 9 nt of RNA, whereas the C-9A mutation resulted in early transition to elongation by synthesis of 7–8 nt of RNA. The effect of early promoter clearance in the mutant promoters was observed as reduced production of long abortive products. [Copyright &y& Elsevier]

Nanostructured ZnO thin film on a glass slide has been prepared by the spin-coating method together with calcining at 500 °C for 2 h in flowing oxygen atmosphere. The grain size of the ZnO nanoparticles is estimated to be ca. 12 nm as determined by the absorption spectrum and powder X-ray diffraction analysis. The photoluminescent peaks that centered at ca. 379 and 388 nm are assigned to the spontaneous emission and exciton–exciton emission, respectively. The thin film also shows frequency-tripled properties for the output when the laser beam of 1.06 μm is input in convergent beam. [Copyright &y& Elsevier]

Hypocrellin A (HA) is encapsulated in the mesoporous material MCM-41 for the first time, and its excited-state behaviors are elucidated by means of steady-state spectroscopies and excited-state lifetime measurements. No ground-state tautomer of HA in the mesoporous solid is deduced based on the spectra observed. Normal fluorescence energy level results from the (l, aπ) state with pπ conjugation; the excited-state intramolecular proton transfer (ESIPT) fluorescence is mostly due to the two-photon excitation (or absorption) of HA. The ESIPT emission intensity becomes truly remarkable with increasing excitation laser intensities. The time-resolved fluorescence measurements are also performed as the further evidence. [Copyright &y& Elsevier]

The photoluminescence behavior of ZnO is largely dependent on its defect properties that are resulted from different preparation conditions. In this Letter, several kinds of nanosized ZnO are successfully encapsulated in the mesopores of the MCM-41 with different starting materials and at various calcination atmospheres. A novel long-lifetime photoluminescence of the nanostructured ZnO is observed for the first time and is attributed to the donor–acceptor pair (DAP) luminescence. The time-delayed spectra and decay kinetics of the DAP luminescence are also presented. An energy-state model depicting the luminescence is proposed finally, which would be useful for our understanding the mechanism of the blue or green emission of ZnO. [Copyright &y& Elsevier]

Methylene blue (MB) is a phenothiazinium photosensitizer with promising applications in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to herring sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the elucidation of the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern–Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the Kb value of 1.89 × 104 M-1. The photobleaching processes of MB and its DNA complex have also been studies, which indicate that the photobleaching of MB and its DNA complex proceeds with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB. [Copyright &y& Elsevier]

Summary Cytosolic pyruvate kinase (ATP:pyruvate 2-O -phosphotransferase, EC 2.7.1.40) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220 DLVN) and site-2 (VLTRGGS407 TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc ) is phosphorylated at both sites in vivo . However, by SDS–PAGE, the phosphorylated polypeptides were found to be smaller (20–51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro , the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo . Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level.... [ABSTRACT FROM AUTHOR]

ZnO nanoparticles have been successfully loaded in the all-silica molecular sieve MCM-41. Combined powder X-ray diffraction and infrared spectral measurements show that ZnO particles have been formed and dispersed in the nanoporous channels host. The marked blue shifts in the electronic absorption band and visible fluorescence are ascribed to the quantum size effects. The visible photoluminescence is attributed to the defects that are related to the anionic oxygen vacancies. [Copyright &y& Elsevier]

An organic molecule salicylidene-1,2-ethanediamine 1, has been encapsulated in the nanocavities of MCM-41 and this nanocomposite material has been investigated by X-ray diffraction, absorption and emission spectroscopy. Results from the spectroscopic measurements show that the bathochromic shift of the 0–0 transitions is correlated with the reduction of the HOMO–LUMO band gap accompanying by the energy changes of the frontier orbitals. Theoretical studies indicate that the energy levels of HOMO and LUMO increase when 1 is confined, and the HOMO is more sensitive than the LUMO. [Copyright &y& Elsevier]

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague-Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI. [ABSTRACT FROM AUTHOR]

Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5?-triphosphate (ppp), by single-stranded RNA marked by a 5?-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5?-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM. [ABSTRACT FROM AUTHOR]

Since the publication of our article [1] it has come to our attention that there was an error in Figure 4 in which the bottom left immunochemistry panel Control/Bax was a duplication of the bottom right immunohistochemistry panel EGCG/GDNF in Figure 3. [ABSTRACT FROM AUTHOR]

A new Shiff base molecule N,N′-bis(2-hydroxy-5-methylbenzylidene)-1,2-ethanediamine (1) has been successfully encapsulated in the nanoporous channels of MCM-41 host. The bathochromic shifts of the 0–0 transitions have been correlated with the reduction of the HOMO–LUMO band gap accompanying by the increased energies of the frontier orbitals. This trend indicates that the molecular orbital confinement of organic molecules is indeed realized in larger cavities, such as nanoporous MCM-41. The variations in the 0–0 transitions observed here have also been confirmed by theoretical calculations. [Copyright &y& Elsevier]

A novel heteronuclear compound, [Na8Zn4(CH3CO2)16·2H2O]n 1, with an unusual two-dimensional condensed polymeric structure was successfully synthesized. 1 crystallizes in the triclinic space group P-1, a=9.3332(19) A˚, b=10.511(2) A˚, c=16.152(3) A˚, α=79.78(3)°, β=83.82(3)°, γ=64.85(3)°, V=1410.6(5) A˚3, Z=2. The most striking feature of 1 is that the bridging acetate ligands show four coordination modes within one compound. The luminescence studies show strong blue photoluminescence as the result of the fluorescence from the intraligand emission excited state. [Copyright &y& Elsevier]