Contribution Of Residues Research Articles

Design and characterization of hyper stable variant of human fibroblast growth factor1 (hFGF1).

Human Fibroblast growth factor 1 (hFGF1) is a powerful signaling molecule belongs to FGF1 subfamily and a key member of biological process related to cell development and wound healing. Studies have revealed that under physiological temperatures, hFGF1 has low thermodynamic stability and biological activity. The Cysteine residues present in hFGF1 have a prominent effect on folding equilibrium of the protein. Studies to determine the contribution of these invariant cysteine residues to the inherent stability of the hFGF1 can lead to potential therapeutic application in the field of wound healing. Interestingly, mutational substitution of Cystein117 residue with alanine in hFGF1 has proven to govern the inherent stability and biological activity results. Detailed analysis of the results will be presented.

Effect of alanine versus serine at position 88 of human transthyretin mutants on the protein stability.

Human transthyretin (TTR) is a homo-tetrameric plasma protein associated with a high percentage of β-sheet forming amyloid fibrils. It accumulates in tissues or extracellular matrices to cause amyloid diseases. Free energy simulations with thermodynamic integration based on all-atom molecular dynamics simulations have been carried out to analyze the effects of the His88→Ala and Ser mutations on the stability of human TTR. The calculated free energy change differences (ΔΔG) caused by the His88→Ala and His88→Ser mutations are -1.84±0.86 and 7.56±0.55kcal/mol, respectively, which are in excellent agreement with prior reported experimental values. The simulation results show that the H88A mutant is more stable than the wild type, whereas the H88S mutant is less stable than the wild type. The free energy component analysis shows that the contribution to the free energy change difference (ΔΔG) for the His88→Ala and His88→Ser mutations mainly arise from electrostatic and van der Waals interactions, respectively. The electrostatic term stabilizes the H88A mutant more than the wild type, but the van der Waals interaction destabilizes the H88S mutant relative to the wild type. Individual residue contributions to the free energy change show neighboring residues exert stabilizing and destabilizing influence on the mutants. The implications of the simulation results for understanding the stabilizing and destabilizing effect and its contribution to protein stability are discussed.

Selective mechanism of inhibitors to two bromodomains of BRD4 revealed by multiple replica molecular dynamics simulations and free energy analyses

Bromodomain-containing protein 4 (BRD4) is critical in cell cycle regulation and has emerged as a potential target for treatment of various cancers. BRD4 contains two bromodomains, namely BDl and BD2. Research suggests that selectively inhibiting BDl or BD2 may provide more effective treatment options. Therefore, understanding the selective mechanism of inhibitor binding to BDl and BD2 is essential for development of high selective inhibitors to BDl and BD2. Multiple replica molecular dynamics (MRMD) simulations are utilized to investigate the binding selectivity of inhibitors SG3-179, GSK778, and GSK620 for BDl and BD2. The results show that BDl has stronger structural flexibility than BD2, moreover BDl and BD2 exhibit different internal dynamics. The analyses of free energy landscapes reveal significant differences in the conformational distribution of BDl and BD2. Binding free energy predictions suggest that entropy changes, electrostatic interactions, and van der Waals interactions are key factors in the selective binding of BDl and BD2 by SG3-179, GSK778, and GSK620. The calculations of the energy contributions of individual residues demonstrate that residues (W81, W374), (P82, P375), (Q85, K378), (V87, V380), (192, 1385), (N93, G386), (194, 1387), (C136, C429), (N140, N433), (K141, P434), (D144, H437) and (1146, V439) corresponding to (BDl, BD2) generate significant energy difference in binding of SG3-179, GSK778, and GSK620 to BDl and BD2, and they can serve as effective targets for development of high selective inhibitors against BDl or BD2. The related information may provide significant theoretical guidance for improving the selectivity of inhibitors for BDl and BD2.

Structural Dynamics of P‐Rex1 Complexed with Natural Leads Establishes the Protein as an Attractive Target for Therapeutics to Suppress Cancer Metastasis

Phosphatidylinositol 3,4,5‐trisphosphate‐ (PIP3‐) dependent Rac exchanger 1 (P‐Rex1) functions as Rho guanine nucleotide exchange factor and is activated by synergistic activity of Gβγ and PIP3 of the heterotrimeric G protein. P‐Rex1 activates Rac GTPases for regulating cell invasion and migration and promotes metastasis in several human cancers including breast, prostate, and skin cancer. The protein is a promising therapeutic target because of its multifunction roles in human cancers. Herein, the present study attempts to identify selective P‐Rex1 natural inhibitors by targeting PIP3‐binding pocket using large‐size multiple natural molecule libraries. Each library was filtered subsequently in FAF‐Drugs4 based on Lipinski’s rule of five (RO5), toxicity, and filter pan assay interference compounds (PAINS). The output hits were virtually screened at the PIP3‐binding pocket through PyRx AutoDock Vina and cross‐checked by GOLD. The best binders at the PIP3‐binding pocket were prioritized using a comparative analysis of the docking scores. Top‐ranked two compounds with high GOLD fitness score (>80) and lowest AutoDock binding energy (< ‐12.7 kcal/mol) were complexed and deciphered for molecular dynamics along with control‐P‐Rex1 complex to validate compound binding conformation and disclosed binding interaction pattern. Both the systems were seen in good equilibrium, and along the simulation time, the compounds are in strong contact with the P‐Rex1 PIP3‐binding site. Hydrogen bonding analysis towards simulation end identified the formation of 16 and 22 short‐ and long‐distance hydrogen bonds with different percent of occupancy to the PIP3 residues for compound I and compound 2, respectively. Radial distribution function (RDF) analysis of the key hydrogen bonds between the compound and the PIP3 residues demonstrated a strong affinity of the compounds to the mentioned PIP3 pocket. Additionally, MMGB/PBSA energies were performed that confirmed the dominance of Van der Waals energy in complex formation along with favorable contribution from hydrogen bonding. These findings were also cross‐validated by a more robust WaterSwap binding energy predictor, and the results are in good agreement with a strong binding affinity of the compounds for the protein. Lastly, the key contribution of residues in interaction with the compounds was understood by binding free energy decomposition and alanine scanning methods. In short, the results of this study suggest that P‐Rex1 is a good druggable target to suppress cancer metastasis; therefore, the screened druglike molecules of this study need in vitro and in vivo anti‐P‐Rex1 validation and may serve as potent leads to fight cancer.

Cooperation of structural motifs controls drug selectivity in cyclin-dependent kinases: an advanced theoretical analysis

Understanding drug selectivity mechanism is a long-standing issue for helping design drugs with high specificity. Designing drugs targeting cyclin-dependent kinases (CDKs) with high selectivity is challenging because of their highly conserved binding pockets. To reveal the underlying general selectivity mechanism, we carried out comprehensive analyses from both the thermodynamics and kinetics points of view on a representative CDK12 inhibitor. To fully capture the binding features of the drug-target recognition process, we proposed to use kinetic residue energy analysis (KREA) in conjunction with the community network analysis (CNA) to reveal the underlying cooperation effect between individual residues/protein motifs to the binding/dissociating process of the ligand. The general mechanism of drug selectivity in CDKs can be summarized as that the difference of structural cooperation between the ligand and the protein motifs leads to the difference of the energetic contribution of the key residues to the ligand. The proposed mechanisms may be prevalent in drug selectivity issues, and the insights may help design new strategies to overcome/attenuate the drug selectivity associated problems.

Network analysis uncovers the communication structure of SARS-CoV-2 spike protein identifying sites for immunogen design

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to understand the structure and dynamics of this complex pathogen. The spike glycoprotein of SARS-CoV-2 is a significant target for immunogens as it is the means by which the virus enters human cells, while simultaneously sporting mutations responsible for immune escape. These functional and escape processes are regulated by complex molecular-level interactions. Our study presents quantitative insights on domain and residue contributions to allosteric communication, immune evasion, and local- and global-level control of functions through the derivation of a weighted graph representation from all-atom MD simulations. Focusing on the ancestral form and the D614G-variant, we provide evidence of the utility of our approach by guiding the selection of a mutation that alters the spike's stability. Taken together, the network approach serves as a valuable tool to evaluate communication "hot-spots" in proteins to guide design of stable immunogens.

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

Effect of Double Mutation (L452R and E484Q) on the Binding Affinity of Monoclonal Antibodies (mAbs) against the RBD-A Target for Vaccine Development.

The COVID-19 pandemic, caused by SARS-CoV-2, emerges as a global health problem, as the viral genome is evolving rapidly to form several variants. Advancement and progress in the development of effective vaccines and neutralizing monoclonal antibodies are promising to combat viral infections. In the current scenario, several lineages containing "co-mutations" in the receptor-binding domain (RBD) region of the spike (S) protein are imposing new challenges. Co-occurrence of some co-mutations includes delta (L452R/T478K), kappa (L452R/E484Q), and a common mutation in both beta and gamma variants (E484K/N501Y). The effect of co-mutants (L452R/E484Q) on human angiotensin-converting enzyme 2 (hACE2) binding has already been elucidated. Here, for the first time, we investigated the role of these RBD co-mutations (L452R/E484Q) on the binding affinity of mAbs by adopting molecular dynamics (MD) simulation and free-energy binding estimation. The results obtained from our study suggest that the structural and dynamic changes introduced by these co-mutations reduce the binding affinity of the viral S protein to monoclonal antibodies (mAbs). The structural changes imposed by L452R create a charged patch near the interfacial surface that alters the affinity towards mAbs. In E484Q mutation, polar negatively charged E484 helps in the formation of electrostatic interaction, while the neutrally charged Q residue affects the interaction by forming repulsive forces. MD simulations along with molecular mechanics-generalized Born surface area (MMGBSA) studies revealed that the REGN 10933, BD-368-2, and S2M11 complexes have reduced binding affinity towards the double-mutant RBD. This indicates that their mutant (MT) structures have a stronger ability to escape from most antibodies than the wild type (WT). However, EY6A Ab showed higher affinity towards the double MT-RBD complex as compared to the WT. However, no significant effect of the per-residue contribution of double-mutated residues was observed, as this mAb does not interact with the region harboring L452 and E484 residues.

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni)

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Investigation of enhanced CO2 storage in deep saline aquifers by WAG and brine extraction in the Minnelusa sandstone, Wyoming

Geological CO2 sequestration in deep saline aquifers has been extendedly investigated to reach the goal of carbon neutral as large amount of CO2 can be reduced in a short time. This study investigates the feasibility of water-alternating-gas (WAG) injection and brine extraction on enhancing CO2 storage in a target deep saline aquifer, Minnelusa Sandstone in the Powder River Basin (PRB) of Wyoming. An integrated numerical model is developed to account for four scenarios: (1) CO2 continuous injection (CI) through one injection well, (2) WAG injection through one injection well, (3) CO2 CI through one injection well and brine extraction through one producer, (4) WAG injection through one injection well and brine extraction through one producer. Simulation results suggest that WAG injection and brine extraction corporately make it possible to enhance CO2 injectivity while securing CO2 storage safety. For instance, WAG injection considerably reduces structural trapping contribution while enhances dissolved and residual trapping contributions. As for brine extraction, it can decrease the maximum averaged reservoir pressure by 37% and 22% for CI and WAG injection, respectively. Besides, sensitive analyses of the operational parameters for the fourth scenarios are performed. Results reveal that, when the amount of total CO2 injection has been predetermined, CO2 injection time and rate within each period of WAG should be small whereas it is preferable to have large water injection time and rate. This is to secure desirable dissolved and residual trapping contributions (store CO2 with safety). Sensitive degree results confirm that CO2 injection rate, water injection time, and CO2 injection time of WAG injection exhibit significant effects on CO2 storage whereas the impacts of producer bottom-hole pressure (BHP), water injection rate, and CO2-water injection time are relatively weaker. This study not only sheds light on achieving double-win goal: enhance CO2 injectivity and store CO2 with safety, but also can be a critical reference for other CO2 storage in deep saline aquifers.

Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5.

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

Residual Behavior of Tebuconazole and Trifloxystrobin in Okra Fruits and Soil

ABSTRACT A field trial was conducted to study the dissipation of trifloxystrobin and tebuconazole on okra following application of Nativo (tebuconazole 50% + trifloxystrobin 25% WG) at 350 and 700 g ha−1. The estimation of trifloxystrobin and CGA 321113 residues was carried on gas–liquid chromatograph equipped with electron capture detector and that of tebuconazole on gas chromatograph–mass spectrometry. Average initial deposits of tebuconazole in okra were found to be 0.75 and 1.45 mg kg−1, whereas trifloxystrobin was found to be 0.28 and 0.61 mg kg−1 following the application of Nativo at 350 and 700 g ha−1, respectively. Residues of tebuconazole dissipated below limit of quantification of 0.05 mg kg−1 after 7 and 10 days and of trifloxystrobin after 5 days of last application at recommended and double of the recommended dosages, respectively. For risk assessment studies, theoretical maximum residue contributions (TMRC) were calculated and compared with maximum permissible intake (MPI). It was observed that TMRC values were below MPI in zero day samples at both recommended and double of the recommended dosages. Therefore, the waiting period of 1 day should be observed for safe consumption of okra treated with tebuconazole and trifloxystrobin.

Rationally designed antifungal protein chimeras reveal new insights into structure-activity relationship

Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.

Matrix and fabric contribution on the tensile behaviour of fabric reinforced cementitious matrix composites

Fabric reinforced cementitious matrix (FRCM) are a type of sustainable materials for structures strengthening and envisaging to develop new construction solutions. However, the wide range of possible combinations of fabric and cementitious matrixes makes it necessary to study the particular contribution of mortar and fabric in the composite action of the FRCM. In this line, this study presents the experimental, analytical and numerical evaluation of the tensile behaviour of FRCM composites. 85 FRCM specimens of different thicknesses were manufactured, with one or two embedded fabrics of different type of fibres and geometry. Fabrics made of coated vegetal fibres were also considered to be compared with commercial fabrics of coated synthetic fibres. The results show a significant increase of the ultimate tensile strength with the increase of the FRCM thickness due to a residual contribution of the mortar after the cracking stage. The analytical model was effective for the cases that showed a cracking stabilization stage. However, the numerical model allows a closer approximation to experimental results because of considering the mortar contribution in the post-cracking stage.

Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase

The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.

Mean annual temperature and carbon availability respectively controlled the contributions of bacterial and fungal residues to organic carbon accumulation in topsoil across China's forests

AbstractAimSoil organic carbon (SOC) stabilization has become an important topic in recent years in the context of global climate change. Microbial residues represent a significant component of stabilized SOC pools. However, spatial variations in the contributions of bacterial and fungal residues to SOC and their determinants at a continental scale remain poorly understood. We aimed to evaluate the spatial variations and controls of the contributions of microbial residues to SOC in forest topsoil.LocationNorth–south transect in eastern China.Time period2014.Major taxa studiedForest ecosystems.MethodsA total of 195 surface (0–10 cm) soils were sampled from 28 forest sites across tropical and boreal forests in eastern China from July to August to assess how biotic and abiotic factors govern the geographic patterns of the contributions of soil microbial residues (indicated by amino sugars) to SOC.ResultsFungal residues (30.0%) had a greater average contribution to SOC than bacterial residues (15.5%). The contributions of bacterial (CBR) and total microbial residues (CMR) to SOC showed negative latitudinal patterns and were positively correlated with mean annual temperature (MAT). In contrast, the contribution of fungal residues to SOC (CFR) showed no clear geographic or climatic patterns. On average, the CBR (9.7%), CFR (21.1%) and CMR (30.8%) were lower in boreal forests than in other biome forests. SOC concentration negatively mediated CBR, CFR and CMR. The piecewise structural equation model results showed that MAT was the primary driver of the geographic pattern of CBR, whereas SOC and soil carbon : nitrogen ratio were more directly associated with the CFR. Additionally, plant factors and microbial properties (i.e., microbial biomass and composition) played relatively little roles in regulating CBR, CFR and CMR.Main conclusionsThese findings advance the current knowledge of the different geographic patterns of CBR and CFR regulated by different potential mechanisms in forest ecosystems. This highlights that the dynamics of microbial residues could potentially have unexpected consequences for topsoil SOC stocks under climate change.

Logistic Regression-Guided Identification of Cofactor Specificity-Contributing Residues in Enzyme with Sequence Datasets Partitioned by Catalytic Properties.

Changing the substrate/cofactor specificity of an enzyme requires multiple mutations at spatially adjacent positions around the substrate pocket. However, this is challenging when solely based on crystal structure information because enzymes undergo dynamic conformational changes during the reaction process. Herein, we proposed a method for estimating the contribution of each amino acid residue to substrate specificity by deploying a phylogenetic analysis with logistic regression. Since this method can estimate the candidate amino acids for mutation by ranking, it is readable and can be used in protein engineering. We demonstrated our concept using redox cofactor conversion of the Escherichia coli malic enzyme as a model, which still lacks crystal structure elucidation. The use of logistic regression with amino acid sequences classified by cofactor specificity showed that the NADP+-dependent malic enzyme completely switched cofactor specificity to NAD+ dependence without the need for a practical screening step. The model showed that surrounding residues made a greater contribution to cofactor specificity than those in the interior of the substrate pocket. These residues might be difficult to identify from crystal structure observations. We show that a highly accurate and inferential machine learning model was obtained using amino acid sequences of structurally homologous and functionally distinct enzymes as input data.

X-ray emission from cosmic web filaments in SRG/eROSITA data

Using the publicly available eROSITA Final Equatorial Depth Survey (eFEDS) data, we detected the stacked X-ray emissions at the position of 463 filaments at a significance of 3.8σbased on the combination of all energy bands. In parallel, we found that the probability of the measurement under the null hypothesis is ∼0.0017. The filaments were identified with galaxies in the Sloan Digital Sky Survey survey, ranging from 30 Mpc to 100 Mpc in length at 0.2 < z < 0.6. The stacking of the filaments was performed with the eFEDS X-ray count-rate maps in the energy range between 0.4 and 2.3 keV after masking the resolved galaxy groups and clusters and the identified X-ray point sources from the ROSAT,Chandra,XMM-Newton, and eROSITA observations. In addition, diffuse X-ray foreground and background emissions or any residual contribution were removed by subtracting the signal in the region between 10 and 20 Mpc from the filament spines. For the stacked signal, we performed an X-ray spectral analysis, which indicated that the signal is associated with a thermal emission. According to a model with the astrophysical plasma emission code for the plasma emission and with aβ-model gas distribution withβ = 2/3, the detected X-ray signal can be interpreted as emission from hot gas in the filaments with an average gas temperature of 1.0−0.2+0.3keV and a gas overdensity of 21 ± 5 at the center of the filaments.

The contribution of stone tool residues in reconstructing Late Pleistocene hominin stone tool behaviour at Grotta di Fumane, Italy

Understanding the relationship between stone tool technology and Late Pleistocene hominins has been one of the most fundamental questions within the field of human evolution. While this question has traditionally been addressed through the technological and typological study of lithic remains, improvements in detecting and identifying stone tool residues offer new avenues for reconstructing Late Pleistocene stone tool technologies. However, the possible contribution of stone tool residues in the reconstruction of Late Pleistocene stone tool technologies remains unclear due to unsolved methodological issues. The remaining issues include uncertainties concerning the exact role of optical microscopy, the effect of the depositional context on residue preservation and the effect of (post-)excavation treatment. This paper explores the possible contribution of stone tool residues through a large-scale residue analysis of a lithic assemblage from Grotta di Fumane, including stone tools from Middle and Upper Palaeolithic layers. Including different microscopic techniques, lithics from different depositional contexts and treated through different (post-)excavation procedures allowed the verification of the effects of all these variables on the possible contribution of residues for reconstructing Late Pleistocene technologies. The results indicate that all these variables play a role in interpreting stone tool residues and should be considered crucial elements of a residue analysis protocol.

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.

The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.