Solubility Controlling Peptide Tags of Opposite Charges Generate a Bivalent Immune Response Against Dengue ED3 Serotypes 3 and 4.

We previously demonstrated that a protein's immunogenicity could be substantially increased by attaching a hydrophobic solubility controlling peptide tag (SCP-tag) producing small sub-visible aggregates. Here, we report the oligomerization of Dengue envelop protein domain 3 (ED3), and consequently, its immunogenicity increase by mixing ED3s attached with SCP-tags of opposite charges at equimolar concentration. We used ED3 of serotype 3 (D3ED3) and serotype 4 (D4ED3), which are, respectively, moderately and poorly immunogenic, and their SCP tagged variants constructed by attaching either a C-termini 5-Aspartic acid (C5D) or a 5-Lysine (C5K) tag. Light scattering indicated that the isolated tagged ED3s remained monomeric, but mixing the C5D and C5K tagged ED3s at equimolar concentration generated sub-visible aggregates or oligomers of ~500 nm through electrostatic interaction. In addition, the oligomerized ED3s remained in a native-like state, as assessed by fluorescence spectroscopy and circular dichroism. The in vivo immunogenicity of the D3ED3 and D4ED3 oligomers generated by the charged tags increased by 5 and 16 fold, respectively. Furthermore, injection of heterotypic ED3 oligomers (D3C5D+D4C5K) induced an immune response against both D3ED3 and D4ED3 in 3 of 4 responsive mice, and the IgG titer of the bivalent anti-D3C5D-D4C5K sera was over 100 times higher than that generated by co-injecting the untagged D3ED3 and D4ED3 (D3+D4). Altogether, these observations suggest that SCP-tags could be used as a platform for producing a long-sought tetravalent dengue vaccine.

Ivermectin in combination with doxycycline for treating COVID-19 symptoms: a randomized trial.

OBJECTIVE: We evaluated whether ivermectin combined with doxycycline reduced the clinical recovery time in adults with COVID-19 infection. METHODS: This was a randomized, blinded, placebo-controlled trial in patients with mild-to-moderate COVID-19 symptoms randomly assigned to treatment (n = 200) and placebo (n = 200) groups. The primary outcome was duration from treatment to clinical recovery. Secondary outcomes were disease progression and persistent COVID-19 positivity by RT-PCR. RESULTS: Among 556 screened patients, 400 were enrolled and 363 completed follow-up. The mean patient age was 40 years, and 59% were men. The median recovery time was 7 (4-10, treatment group) and 9 (5-12, placebo group) days (hazard ratio, 0.73; 95% confidence interval, 0.60-0.90). The number of patients with a ≤7-day recovery was 61% (treatment group) and 44% (placebo groups) (hazard ratio, 0.06; 95% confidence interval, 0.04-0.09). The proportion of patients who remained RT-PCR positive on day 14 and whose disease did not progress was significantly lower in the treatment group than in the placebo group. CONCLUSIONS: Patients with mild-to-moderate COVID-19 infection treated with ivermectin plus doxycycline recovered earlier, were less likely to progress to more serious disease, and were more likely to be COVID-19 negative by RT-PCR on day 14. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04523831. DATA REPOSITORY ID: Dryad. doi:10.5061/dryad.qjq2bvqf6.

A systematic mutational analysis identifies a 5-residue proline tag that enhances the in vivo immunogenicity of a non-immunogenic model protein.

Poor immunogenicity of small proteins is a major hurdle in developing vaccines or producing antibodies for biopharmaceutical usage. Here, we systematically analyzed the effects of 10 solubility controlling peptide tags (SCP-tags) on the immunogenicity of a non-immunogenic model protein, bovine pancreatic trypsin inhibitor (BPTI-19A; 6 kDa). CD, fluorescence, DLS, SLS, and AUC measurements indicated that the SCP-tags did not change the secondary structure content nor the tertiary structures of the protein nor its monomeric state. ELISA results indicated that the 5-proline (C5P) and 5-arginine (C5R) tags unexpectedly increased the IgG level of BPTI-19A by 240- and 73-fold, respectively, suggesting that non-oligomerizing SCP-tags may provide a novel method for increasing the immunogenicity of a protein in a highly specific manner.

Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein.

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags.

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25°C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25°C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37°C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.

Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

Prevalence and assessment of biochemical parameters of attention-deficit hyperactivity disorder children in Bangladesh.

BACKGROUND AND OBJECTIVES: This study aimed to evaluate some new biochemical parameters that help ensuring the early and precise diagnosis of attention-deficit hyperactivity disorder (ADHD) in blood plasma. DESIGN AND SETTINGS: A prospective study conducted with patients scheduled for some new biochemical parameters that help ensuring the early and precise diagnosis of ADHD in blood plasma in a Child Development Center of the Chittagong, Bangladesh. MATERIALS AND METHODS: The study was carried out at two levels. The first level was questionnaire on personal data and disease history while the second was on biochemical examination of the plasma ammonia and lactate status. A total of 100 children (age range 2 years 4 months to 12 years 6 months, mean age 7 years 5 months) were investigated in this study among 75 were male and 25 were female. This study was conducted in Chittagong Maa-O-Shishu General Hospital, Bangladesh. RESULTS: We observed that the level of plasma ammonia and lactate were higher in ADHD children (36-60 µmol/L; P < 0.05 and 22-30 µmol/L; P < 0.05, respectively) compare to a reference value. The prevalence of ADHD is higher in male (75%) than in female (25%) with a ratio of 3:1. Consanguinity increases the risk of having ADHD in the next generation. CONCLUSION: This study concludes that there might be a correlation between ADHD and increased level of plasma ammonia and lactate level, and those might be an important parameter in the diagnosis of ADHD patients.

Analysis of protein aggregation kinetics using short amino acid peptide tags.

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10mg/ml to 25.0mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the "transient solubility (TS)", the "aggregation initiation concentration (AIC)" and the "long-term solubility (LS)". AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the "solubility" of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.

Analysis of amino acid contributions to protein solubility using short peptide tags fused to a simplified BPTI variant.

Protein solubility is usually characterized in terms of a hydrophobicity scale, which refers to the free energy of transfer of a molecule from an aqueous to a nonpolar solution and is not a "solubility propensity scale" per se. Using a "host-guest" approach, we measured the effects of short poly-amino-acid tags (guests) on the solubility of a host protein, a simplified bovine pancreatic trypsin inhibitor (BPTI), to which they were fused at the C-terminus. We analyzed 10 amino acid types, representing the full range of biophysical properties (acidic, basic, polar, and hydrophobic). As anticipated, positively charged residues significantly increased the solubility of the model protein, at both pH 4.7 and 7.7, whereas very hydrophobic poly-Ile markedly reduced the solubility of BPTI. Poly-Asp and poly-Glu barely affected BPTI solubility at pH 4.7, but induced an eight to ten-fold increase at pH 7.7, attributable to the ionization of their side chains. Although Pro is the most soluble amino acid, poly-Pro did not affect the protein's solubility. The effects of the other tags on BPTI solubility ranged from none to an eight-fold increase. To ensure that the measured solubility values were context independent and could provide a "solubility propensity scale", we confirmed that the tags remained independent of the structure, thermal stability, and biochemical activity of the host protein. These observations suggest that this approach is valuable for measuring the solubility propensities of amino acids, which could eventually allow the calculation of a polypeptide's relative solubility from its amino acid sequence.

Geostatistical assessment of the impact of World War I on the spatial occurrence of soil heavy metals.

Previous research showed a regional Cu enrichment of 6 mg kg(-1) in the top soil of the Ypres war zone (Belgium), caused by corrosion of WWI shell fragments. Further research was required since in addition to Cu, also As, Pb, and Zn were used during the manufacturing of ammunition. Therefore, an additional data collection was conducted in which the initial Cu data set was tripled to 731 data points and extended to eight heavy metals (As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn) which permitted (1) to evaluate the environmental impact of the heavy metals at a regional scale and (2) to assess their regional spatial occurrence by performing an optimized geostatistical modeling. The results showed no pollution at a regional scale, but sometimes locally concentrations exceeded the soil sanitation threshold, especially for Cu, Pb, and Zn. The spatial patterns of Ni and Cr were related to variations in soil texture whereas the occurrences of Cu and Pb were clearly linked to WWI activities. This difference in spatial behavior was confirmed by an analysis of coregionalization.

Thermodynamic and structural analysis of highly stabilized BPTIs by single and double mutations.

Enhancing protein conformational stability is an important aspect of protein engineering and biotechnology. However, protein stabilization is difficult to rationalize as it often results from the small cumulative and intertwined effects of multiple mutations. Here, we analyzed the mechanisms behind a remarkable 13 degrees stabilization produced by a single A14G and a double A14GA38V mutation in BPTI-[5,55], a natively folded bovine pancreatic trypsin inhibitor variant. Differential scanning calorimetry analysis of three BPTI-[5,55] variants (A14G, A38V, and A14GA38V) indicated that the A14G mutation stabilized the structure enthalpically, whereas the A38V stabilization was entropy driven. We also determined the structure of the A14GA38V mutant at 1.09 A resolution, whereas the A38V variant did not crystallize, and we previously reported the A14G variant's structure (2ZJX). The overall structures of the A14G and A14GA38V variants were very similar to that of wild-type BPTI, but small local structure perturbations around residues 14 and 38 strongly suggested potential factors contributing to the enthalpy stabilization. First, the A14G mutation displaced the local backbone structures around residues 14 and 38 by up to 0.7 A, presumably increasing local van der Waals interactions. Next, this displacement produced steric clashes between neighboring residue's side-chains in all but the variants containing the A14G mutation. Noteworthy, these clashes are not predicted from the wild type BPTI structure. These observations provide one of the first unambiguous analyses of how a subtle interplay between the sidechain and backbone structures can have a major effect on protein stability.

Crystal structure of an extensively simplified variant of bovine pancreatic trypsin inhibitor in which over one-third of the residues are alanines.

We report the high-resolution crystal structures of an extensively simplified variant of bovine pancreatic trypsin inhibitor containing 20 alanines (BPTI-20st) and a reference single-disulfide-bonded variant (BPTI-[5,55]st) at, respectively, 1.39 and 1.09 A resolutions. The sequence was simplified based on the results of an alanine scanning experiment, as reported previously. The effects of the multiple alanine substitutions on the overall backbone structure were surprisingly small (C(alpha) atom RMSD of 0.53 A) being limited to small local structural perturbations. Both BPTI variants retained a wild-type level of trypsin inhibitory activity. The side-chain configurations of residues buried in the hydrophobic cores (<30% accessible surface area) were almost perfectly retained in both BPTI-20st and BPTI-[5,55]st, indicating that neither multiple alanine replacements nor the removal of the disulfide bonds affected their precise placements. However, the side chains of three partially buried residues (Q31, R20, and to some extent Y21) and several unburied residues rearranged into alternative dense-packing structures, suggesting some plasticity in their shape complementarity. These results indicate that a protein sequence simplified over its entire length can retain its densely packed, native side-chain structure, and suggest that both the design and fold recognition of natively folded proteins may be easier than previously thought.