Development of a Subclonal host cell line for AAV production

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N protein from lambdoid phages transforms NusA into an antiterminator by modulating NusA-RNA polymerase flap domain interactions

Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of E. coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. The presence of N reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the β-flap as well as the β′ dock domains to form a more constricted RNA exit channel in the N-modified Elongation Complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process

Medial open wedge high tibial osteotomy: an effective procedure for varus malunited neglected proximal tibial plateau fractures: a case series

Varus malunited tibial plateau fracture is a disabling condition causing varus thrust gait, instability, and secondary osteoarthrosis. There are few evidence-based guidelines for the management of symptomatic varus malunion in young patients. Our case series aims to review outcomes of open wedge high tibial osteotomy for varus malunited tibial medial plateau fractures. We present case series of three patients who presented with symptomatic varus malunion of proximal tibia with Kellgren-Lawrence stage II or less arthritic changes. All presented with varus thrust. Exclusion criteria for the procedure would be complex intra-articular step malunions and severe arthritic changes. Medial open wedge high tibial osteotomy was performed and stabilized with locking plate and tricortical autograft. Patients were followed up for 12 to 20 months. All achieved deformity correction, reported significant improvement in varus thrust and were satisfied with their improvement in stability and knee function. There was no complication related to wound healing and no delayed loss of correction. The osteotomy led to correction of the coronal deformity and ensured that the collateral balance is restored benefiting the overall gait of the patient. Also, if such a patient undergoes total knee replacement in future, the bone stock will be significantly better with this procedure. Medial open wedge high tibial osteotomy for varus malunited tibial plateau fractures is effective procedure that provides excellent functional and radiologic outcomes, and carries minimal complications

The interaction surface of a bacterial transcription elongation factor required for complex formation with an antiterminator during transcription antitermination

The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription Elongation Complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA N terminus (NTD) specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53 (S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C) of the NusA binding region of the N protein. We conclude that when bound to the EC, the N interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site) and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals

Cosmic optical activity from an inhomogeneous Kalb-Ramond field

The effects of introducing a harmonic spatial inhomogeneity into the Kalb-Ramond field, interacting with the Maxwell field according to a `string-inspired' proposal made in earlier work are investigated. We examine in particular the effects on the polarization of synchrotron radiation from cosmologically distant (i.e. of redshift greater than 2) galaxies, as well as the relation between the electric and magnetic components of the radiation field. The rotation of the polarization plane of linearly polarized radiation is seen to acquire an additional contribution proportional to the square of the frequency of the dual Kalb-Ramond axion wave, assuming that it is far smaller compared to the frequency of the radiation field.Comment: 9 pages, Revtex, no figure

A radiological evaluation of loop length change in adjustable versus fixed loop femoral cortical fixation devices in arthroscopic anterior cruciate ligament reconstruction: a prospective study

Background: In anterior cruciate ligament reconstructions, fixed devices require over-drilling to flip the button whereas loops of adjustable devices can be adjusted intraoperatively, and they minimize over-drilling. But they can loosen rendering the reconstruction incompetent. Most studies comparing them are bio-mechanical studies. Our aim was to record and compare loop length change radiologically in adjustable versus fixed devices in clinical settings. Methods: 32 patients were divided into 2 groups of 16 patients each. Hamstring graft were prepared. It was loaded in the suspension device and the apex of the graft was marked using silicon vascular radio-opaque marker. In adjustable devices, lengthening was checked after cycling and re-tensioning was done intra-operatively. Post-surgery, digital X-ray of the knee was taken in true antero-posterior and lateral view. Distance between the centre point of the button and the centre-point of the radio-opaque inert silicon marker was recorded at immediate post-operative and at 6 weeks respectively and compared. Results: 15 patients in each group were incorporated. Intra-operatively, loop lengthening was seen in all 15 patients with adjustable loop and re-tensioning was done. 2 of the 15 cases showed evidence of radiological loop lengthening however in both cases the lengthening was less than 3 mm and thus was not significant. Conclusions: We in in vivo radiology based clinical study did not find any significant loop lengthening in patients with adjustable loop devices. Hence fixed and adjustable loop devices are comparable