Design and Fabrication of Pneumatically Actuated Valveless Pumps

In this study, a valveless pump was successfully designed and fabricated for the purpose of medium transportation. Different from traditional pumps, the newly designed pump utilizes an actuated or a deflected membrane, and it serves as the function of a check valve at the same time. For achieving the valveless property, an inlet or outlet port positioned in an upper- or lower-layer thin membrane was designed to be connected to an entrance or exit channel. Theoretical analysis and numerical simulation were conducted simultaneously to investigate the large deformation characteristics of the membranes and to determine the proper location of the inlet or outlet port on the proposed pump. Then, the valveless pump was fabricated on the basis of the proposed design. In the experiment, the maximum flow rate of the proposed pump exceeded 12.47 mL/min at a driving frequency of 5.0 Hz and driving pressure of 68.95 kPa.

Influence of the ECAP Tool Channel Geometry on the Structure and Properties of Al-3%Mg Aluminium Alloy

In this study, commercial Al-3%Mg aluminium alloy was subjected to ECAP processing using two different ECAP die configurations. The first one – conventional and the second one modified in which a part of the exit channel in the ECAP die, causes twist deformation, to impose extra shear strains to the sample. The local changes in microstructure were characterized by Light Microscopy, SEM equipped with an EBSD facility and TEM. Mechanical properties of the ECAP processed samples were compared based on hardness measurement. The results showed that when ECAP with modified die, the greater grain and crystalline refinement is possible. The microstructures exhibit high dislocation density within subgrains with non-equilibrium and Moiré boundaries. Moreover, the mechanical examinations display a significant improvement in hardness and calculated yield strength when the ECAP process is conducted using a modified die.

Numerical investigation of flow field around T-shaped spur dyke in a reverse-meandering channel

In this paper the flow characteristics near around T-shape spur dyke situated in reverse meandering channel having rigid bed is simulated using Renormalization Group (RNG) turbulence model with an ANSYS 2018 Fluent software. To solve the model in 3D ways we used Navier-Stroke's equation based on principle of conservation of mass and momentum within a moving fluid. For studying the flow characteristics, Computational Fluid Dynamics ware applied with all geometric parameter and the turbulence was simulated using (RNG) equations of model. In this simulation the structured meshes are used with different diameter and diameter of meshes is high at exit channel for obtaining accuracy in result. In this study we mainly focus on effect of Froude number on flow pattern and several other characteristics like velocity distribution, flow separation, bed shear stress distribution. The final result of this research work is compare with the condition when no structure is present in the channel.

Region 4 of the RNA polymerase σ subunit counteracts pausing during initial transcription

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This “RNAP boosting” activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24 nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IV’s largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IV’s RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs.Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IV’s largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2’s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

Structure of a human 48S translational initiation complex

A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5′ end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo–electron microscopy to determine the structure of a reconstituted human 48S. The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S. The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being “pulled” through the 40S subunit during scanning.

Structural basis of transcription-translation coupling and collision in bacteria

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The lead ribosome potentially contacts RNA polymerase (RNAP) and forms a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here, we present a series of structures representing uncoupled, coupled, and collided expressome states determined by cryo–electron microscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, which stabilizes an otherwise-variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance channel to the RNAP exit channel. In this collided complex, NusG linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.

Anti-pausing activity of region 4 of the RNA polymerase σ subunit and its regulation by σ-remodeling factors

ABSTRACTThe basal transcription factors of cellular RNA polymerases (RNAPs) stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This “RNAP boosting” activity of the initiation factor is shaped by the the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

Structural basis of transcription-translation coupling and collision in bacteria

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit-channel. In this collided complex, NusG-linkage is no longer possible.These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.One Sentence SummaryStructures of the molecular assembly executing gene expression shed light on transcription translation coupling.