Site Saturation Research Articles

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.

Elucidating Residue-Level Determinants Affecting Dimerization of Ebola Virus Matrix Protein Using High-Throughput Site Saturation Mutagenesis and Biophysical Approaches.

The Ebola virus (EBOV) is a filamentous virus that acquires its lipid envelope from the plasma membrane of the host cell it infects. EBOV assembly and budding from the host cell plasma membrane are mediated by a peripheral protein, known as the matrix protein VP40. VP40 is a 326 amino acid protein with two domains that are loosely linked. The VP40 N-terminal domain (NTD) contains a hydrophobic α-helix, which mediates VP40 dimerization. The VP40 C-terminal domain has a cationic patch, which mediates interactions with anionic lipids and a hydrophobic region that mediates VP40 dimer-dimer interactions. The VP40 dimer is necessary for trafficking to the plasma membrane inner leaflet and interactions with anionic lipids to mediate the VP40 assembly and oligomerization. Despite significant structural information available on the VP40 dimer structure, little is known on how the VP40 dimer is stabilized and how residues outside the NTD hydrophobic portion of the α-helical dimer interface contribute to dimer stability. To better understand how VP40 dimer stability is maintained, we performed computational studies using per-residue energy decomposition and site saturation mutagenesis. These studies revealed a number of novel keystone residues for VP40 dimer stability just adjacent to the α-helical dimer interface as well as distant residues in the VP40 CTD that can stabilize the VP40 dimer form. Experimental studies with representative VP40 mutants in vitro and in cells were performed to test computational predictions that reveal residues that alter VP40 dimer stability. Taken together, these studies provide important biophysical insights into VP40 dimerization and may be useful in strategies to weaken or alter the VP40 dimer structure as a means of inhibiting the EBOV assembly.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

Approaching Theoretical Performances of Electrocatalytic Hydrogen Peroxide Generation by Cobalt‐Nitrogen Moieties

AbstractElectrocatalytic oxygen reduction reaction (ORR) has been intensively studied for environmentally benign applications. However, insufficient understanding of ORR 2 e−‐pathway mechanism at the atomic level inhibits rational design of catalysts with both high activity and selectivity, causing concerns including catalyst degradation due to Fenton reaction or poor efficiency of H2O2 electrosynthesis. Herein we show that the generally accepted ORR electrocatalyst design based on a Sabatier volcano plot argument optimises activity but is unable to account for the 2 e−‐pathway selectivity. Through electrochemical and operando spectroscopic studies on a series of CoNx/carbon nanotube hybrids, a construction‐driven approach based on an extended “dynamic active site saturation” model that aims to create the maximum number of 2 e− ORR sites by directing the secondary ORR electron transfer towards the 2 e− intermediate is proven to be attainable by manipulating O2 hydrogenation kinetics.

Approaching Theoretical Performances of Electrocatalytic Hydrogen Peroxide Generation by Cobalt-Nitrogen Moieties.

Electrocatalytic oxygen reduction reaction (ORR) has been intensively studied for environmentally benign applications. However, insufficient understanding of ORR 2 e- -pathway mechanism at the atomic level inhibits rational design of catalysts with both high activity and selectivity, causing concerns including catalyst degradation due to Fenton reaction or poor efficiency of H2 O2 electrosynthesis. Herein we show that the generally accepted ORR electrocatalyst design based on a Sabatier volcano plot argument optimises activity but is unable to account for the 2 e- -pathway selectivity. Through electrochemical and operando spectroscopic studies on a series of CoNx /carbon nanotube hybrids, a construction-driven approach based on an extended "dynamic active site saturation" model that aims to create the maximum number of 2 e- ORR sites by directing the secondary ORR electron transfer towards the 2 e- intermediate is proven to be attainable by manipulating O2 hydrogenation kinetics.

Saturation Effect on the Coherency Loss of Spatially Varying Vertical Ground Motions

The effect of the influence regularity of site saturation and water level on the coherency loss between spatially varying earthquake ground motions (SVEGMs) in a vertical direction is not yet clear. Therefore, taking an onshore-offshore site as an example, the influence regularity of site saturation and water level on the coherency loss of SVEGMs in a vertical direction was studied, in which the saturation degree of the whole site (SD), the groundwater level (GWL), and the thickness of the surface water layer (WLT) were considered. Under each case mentioned above, a large number of vertical in-plane SVEGMs were synthesized and then correspondingly, the mean lagged coherency loss and phase angle of coherence function were obtained, and a series of comparisons were carried out to shed light on the varying trend of the value of lagged coherency loss and phase angle, with predefined parameters. The results showed that SD, GWL, and WLT possess significant effects on the varying trend of the coherence function of vertical SVEGMs.

Non-isothermal crystallization kinetics of CaO-SiO2-B2O3-TiO2 glass studied by differential thermal analysis in the case of “site saturation”

Crystallization characteristics of the CaO-SiO2-B2O3-TiO2 glass have been studied using scanning electron microscopy, X-ray diffraction and differential thermal analysis techniques. The crystallization kinetic parameters of this glass were estimated under non-isothermal conditions using the Matusuta-Sakka equation. Glass with fine particles (<74 μm), case referred to as ‘‘site saturation’’, was used to obtain the non-isothermal differential thermal analysis curves. Bulk glass-ceramics with dense and nonporous morphology are obtained after the heating treatment indicating the strong heterogeneous nucleation at the junction of gain boundaries in the case of “site saturation”. As the content of TiO2 exceeds 18%, the predominant crystal phase changes from CaSiO3 to CaTiSiO5, until CaTiSiO5 becomes the only crystal phase of the glass at 26% TiO2. The crystallization activation energies, EG, are in the range of 245–514 kJ/mol. As TiO2 increases from 10% to 18%, crystallization ability of the glass decreases gradually and the crystallization corresponds to one-dimensional growth mechanism or surface crystallization, while as TiO2 further increases from 18% to 26%, not only does the glass show an increasing crystallization ability, but also the crystal growth changes to the two-dimensional growth mechanism. Within the range of 22%–26%, TiO2 plays the role of strengthening the crystallization of the glass by acting as a major component in the glass to form Ti-bearing compound. From the standpoint of glass-ceramics preparation, the glass containing more TiO2 (such as 26%) with CaTiSiO5 as the only crystal phase is favorable in the present study.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Producing 2-O-α-D-glucopyranosyl-L-ascorbic acid by modified cyclodextrin glucosyltransferase and isoamylase

In this study, site saturation mutagenesis was performed on the - 3 (R44, D86, S90, and D192) and - 6 subsite (Y163, G175, G176, and N189) of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase to enhance its specificity for the donor substrate maltodextrin for 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) preparation. The AA-2G yields produced by the mutants S90D, G176H, and S90D/G176H were 181, 171, and 185g/L, respectively. Our previous study found that the mutant K228R/M230L also increased the AA-2G yield. Therefore, the mutants S90D, G176H, S90D/G176H, and K228R/M230L were further used to generate combinatorial mutants. Among these mutants, the highest AA-2G yield (217g/L) was produced by S90D/K228R/M230L with 500g/L maltodextrin as the glucosyl donor, which was 56g/L higher than that produced by wild-type CGTase. In addition, AA-2G was prepared by adding isoamylase to hydrolyze α-1,6 glucosidic linkages in maltodextrin that could not be utilized by CGTase to improve the utilization rate of maltodextrin. The addition of isoamylase reduced the concentration of maltodextrin from 500 to 350g/L, while the AA-2G yield remained high (208g/L). The preparation of AA-2G by complexing isoamylase with mutant S90D/K228R/M230L reduced the maltodextrin concentration by 150g/L, while the AA-2G yield increased by 47g/L than preparation with wild-type CGTase alone, which laid a foundation for the large-scale preparation of AA-2G. KEY POINTS: • Mutants exhibited improved maltodextrin specificity. • Mutant S90D/K228R/M230L produced high yield of AA-2G with maltodextrin as substrate. • AA-2G was first synthesized by a combination of isoamylase and CGTase.

Reshaping the Binding Pocket of Cellobiose 2-Epimerase for Improved Substrate Affinity and Isomerization Activity for Enabling Green Synthesis of Lactulose.

Enzymatic isomerization of lactose into lactulose via cellobiose 2-epimerase (CE) could provide an eco-friendly route for the industrial production of lactulose, a valuable food prebiotic. However, poor substrate affinity for lactose and preference for epimerization over isomerization hinder this application. Previous studies on CE improvement have focused on random mutagenesis or active site rational design; little is known about the relationship between substrate binding and enzyme efficacy, which was hence the subject of this study. First, residues 372W and 308W were identified as key for disaccharide recognition in CEs based on crystal structure alignment of the N-acetyl-glucosamine 2-epimerase superfamily and site-directed mutation. This binding domain was then reshaped through site saturation mutagenesis, resulting in seven mutants with enhanced isomerization activity. The optimal mutant CsCE/Q371E had significantly enhanced substrate affinity (Km, 269.65 mM vs Km, 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original CsCE. Molecular dynamics simulation further revealed that substituting Gln-371 with Glu strengthened the hydrogen-bonding network and altered the active site-substrate interactions, increasing the substrate stability and shifting the catalytic direction. This study uncovered new information about the substrate binding region and its mechanisms and impact on CE catalytic performance, paving the way for potential commercial applications.

Binding pocket stabilization by high-throughput screening of yeast display libraries.

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.

Quantifying the coupled monovalent and divalent ions sorption in dense ion-exchange membranes

As a universal phenomenon in solution-membrane systems, the coupled competitive monovalent and divalent ions sorption significantly impacts the transport process in ion-exchange membranes. Often, the quantitative and qualitative relationships between them are unclear. Here, we present a lattice-based molecular model to capture the interplay effect of ion-pairing and electrostatic interaction on the competitive partitioning process. In particular, we utilize statistical mechanics to formulate how the monovalent and divalent counterions competitively pair with the fixed charge group in the membranes. To this end, the model can quantify (a) the counterions sorption and (b) the coion repulsion within the membranes, which, collectively, allow us to quantify the ion partitioning and the pairing site saturation. To showcase the capability of our model, we compare our results with published experiment data, where we achieve a relatively good agreement between them. We demonstrate that the competitive ion partitioning depends highly upon (i) the entropy change generated by the counterions pairing with the fixed charge group, (ii) the binding energy, and (iii) the osmotic pressure, and (iv) the electrostatic interaction. These different factors can explain the favorable sorption of divalent counterions in the cation-exchange membranes but monovalent counterions in the anion ones, as previously reported. Altogether, our theoretical approach provides an insight into the fundamental understanding of coupled competitive monovalent and divalent ions sorption within the dense ion-exchange membranes, further enhancing the knowledge of the competitive ion partitioning process.

Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68.

Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding temperature (Tm) of 88°C (361K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the effects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specificity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding affinity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufficient changes in the substrate binding pocket of the enzyme that can efficiently change substrate specificity of the enzyme.

Facile Fabrication of α-Alumina Hollow Fiber-Supported ZIF-8 Membrane Module and Impurity Effects on Propylene Separation Performance.

The separation of C3 olefin and paraffin, which is essential for the production of propylene, can be facilitated by the ZIF-8 membrane. However, the commercial application of the membrane has not yet been achieved because the fabrication process does not meet industrial regulatory criteria. In this work, we provide a straightforward and cost-effective membrane fabrication technique that permits the rapid synthesis of ZIF-8 hollow fiber membranes. The scalability of the technology was confirmed by the incorporation of three ZIF-8 hollow fiber membranes into a single module using an introduced fiber mounting methodology. The molecular sieving characteristics of the ZIF-8 membrane module on a binary combination of C3 olefin and paraffin (C3H6/C3H8 selectivity of 110 and a C3H6 permeance of 13 GPU) were examined at atmospheric conditions. In addition, the high-pressure performance of these membranes was demonstrated at a 5 bar of equimolar binary feed pressure with a C3H6/C3H8 selectivity of 55 and a C3H6 permeance of 9 GPU due to propylene adsorption site saturation. To further accurately portray the separation performance of the membrane on an actual industrial feed, the effect of impurities (ethylene, ethane, butylene, i-butane, and n-butane), which can be found in C3 splitters, was investigated and a considerable decrement (~15%) in the propylene permeance upon an interaction with C4 hydrocarbons was confirmed. Finally, the long-term stability of the ZIF-8 membrane was confirmed by continuous operation for almost a month without any loss of its initial performance (C3H6/C3H8 separation factor of 110 and a C3H6 permeance of 13 GPU). From an industrial point of view, this straightforward technique could offer a number of merits such as a short synthesis time, minimal chemical requirements, and excellent reproductivity.

Effects of Vanadium on the Kinetics of Austenitization from Pearlite in Fe–V–C Alloys

Vanadium effects on the kinetic characteristics of the austenit transformation in Fe-V-C ternary alloys were investigated by microstructure analysis, differential scanning calorimetry (DSC) and the Johnson-Mehl-Avrami-Kolmogorov model (JMAK). The results show that the vanadium gives rise to the refinement of pearlite lamellar spacing before transformation, the original austenite grain size decreases after transformation, and and the increase of the amount of carbide at the grain boundarsy. Studies on kinetics showed that the austenization of the Fe-V-C ternary alloys is characterized by JMAK model involving site saturation nucleation, diffusion-controlled growth, and impingement correction. V increases the diffusion activation energy and decreases the pre-exponential factor for diffusion.

Stabilizing proteins, simplified: A Rosetta-based webtool for predicting favorable mutations.

Many proteins have low thermodynamic stability, which can lead to low expression yields and limit functionality in research, industrial and clinical settings. This article introduces two, web-based tools that use the high-resolution structure of a protein along with the Rosetta molecular modeling program to predict stabilizing mutations. The protocols were recently applied to three genetically and structurally distinct proteins and successfully predicted mutations that improved thermal stability and/or protein yield. In all three cases, combining the stabilizing mutations raised the protein unfolding temperatures by more than 20°C. The first protocol evaluates point mutations and can generate a site saturation mutagenesis heatmap. The second identifies mutation clusters around user-defined positions. Both applications only require a protein structure and are particularly valuable when a deep multiple sequence alignment is not available. These tools were created to simplify protein engineering and enable research that would otherwise be infeasible due to poor expression and stability of the native molecule.

Deconvolution of main hydration kinetic peaks in properly sulfated Portland cements with boundary nucleation and growth models and relation to early-age concrete strength development

When the hydration kinetics of properly sulfated Portland cements is monitored by heat flow calorimetry, the initial dissolution peak and the dormant period is followed by the main hydration peak – formation of calcium-silicate-hydrates and portlandite, the so-called silicate peak. This main peak is superimposed by a peak associated to further tricalcium aluminate dissolution and a surge in precipitation of sulfoaluminates (the so-called sulfate depletion peak) right after sulfate added for setting control is depleted in the material system. In this paper strategies for the deconvolution of silicate peak and sulfate depletion peak are presented. Hereby, the boundary nucleation and growth model and the classical bulk nucleation and growth model serve as tools for deconvolution of calorimetric data. We discuss obtained kinetic parameters and their temperature dependency. The boundary nucleation and growth model for constant nucleation rate emerged as the most suitable model for the silicate reaction, the bulk nucleation and growth model with an order of three, indicating site saturation, as adequate for the sulfate depletion peak. The reaction enthalpy for tricalcium silicate hydration was obtained as 503 J/g, remarkably close to literature values. Calorimetric data related to the sulfate depletion peak is corroborated by X-ray diffraction analysis of formation history of crystalline hydration products. Furthermore, the history of the (silicate) degree of reaction as predicted by the boundary nucleation and growth model for different temperature histories is related to experimentally obtained early-age strength histories. • Investigation of hydration kinetics of commercial, well-sulfated Portland cement. • Main peak (C-S-H and portlandite formation) superimposed by sulfate depletion peak. • Strategies for deconvolution of peaks with Avrami–Cahn models as tools for analysis. • Degree of silicate reaction history is linked to early-age strength gain.

Evolution-Guided Biosynthesis of Terpenoid Inhibitors.

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objective─a transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in Escherichia coli (E. coli)─to evolve a terpene synthase to produce enzyme inhibitors. Site saturation mutagenesis of poorly conserved residues on γ-humulene synthase (GHS), a promicuous enzyme, yielded mutants that improved fitness (i.e., the antibiotic resistance of E. coli) by reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly, a combination of two mutations enhanced the titer of a minority product─a terpene alcohol that inhibits PTP1B─by over 50-fold, and a comparison of similar mutants enabled the identification of a site where mutations permit efficient hydroxylation. Findings suggest that the plasticity of terpene synthases enables an efficient sampling of structurally distinct starting points for building new functional molecules and provide an experimental framework for exploiting this plasticity in activity-guided screens.

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids.

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L482F) or cleavage and polyadenylation specificity factor 3 (N232H), in the Trypanosoma cruzi proteasome β5 subunit (G208S), or in Leishmania donovani CRK12 (G572D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G492 in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G492 edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy.

Padded Dressing with Lidocaine HCL for Reducing Pain during Intravenous Cannulation in Adult Patients: A Randomized Controlled Clinical Trial.

Objective In this study, we aimed to evaluate the effect of administering lidocaine as a local anesthetic at the puncture site prior cannulation on reduction of pain during intravenous cannulation (IVC). Methods A total of 77 patients were divided into two groups as the patients who received a local anesthetic prior IVC procedure (n = 40) and the control group (n = 37). Patients' demographic data, including age, gender, height, weight and body mass index, IV gauge, IV site, heart rate (HR), and oxygen saturation (SpO2) were recorded and analyzed. Patients in both groups scored the pain they felt during IVC through the visual analog scale (VAS) and the verbal descriptor scale (VDS). Results No statistically significant difference was found between the two groups in terms of the demographic features. There was no significant difference between the two groups in terms of the cannula gauges and site of IVC. The mean post-IVC HR value was statistically significantly higher compared to pre-IVC in the control group (p = 0.032), while no difference was found between the mean pre- and postprocedure HR in the lidocaine group. The mean VAS score was significantly lower in the lidocaine group compared to the control group (p < 0.001). There was a significant difference between the groups in terms of the current VDSs. The rate of the patients reporting mild pain was statistically significantly higher in the lidocaine group compared to the control subjects (p < 0.001). Conclusion According to the results of this study, lidocaine HCL-impregnated padded dressing prior IV cannulation significantly reduced pain sensation during IVC.