Ultrathin, Biomimetic, Superhydrophilic Layers of Cross-Linked Poly(phosphobetaine) on Polyethylene by Photografting

Ultrathin, biomimetic, superhydrophilic hydrogel layers, composed of cross-linked poly­(2-methacryloyloxyethyl phosphorylcholine), are formed on low-density polyethylene films via ultraviolet-initiated surface graft polymerization. The layers are 19–58 nm thick as revealed by electron microscopy and have three-dimensional networks; the unique network structure, along with its zwitterionic nature, rather than surface roughness results in superhydrophilicity, that is, the water contact angle around 5°. This superhydrophilicity depends on a variety of factors, including the concentration of the monomer and cross-linker, the type of reaction solvents, the reaction and drying time, the intensity of UV light, and the way of measurement of water contact angles. Superhydrophilicity is obtained under a fixed ratio (e.g., 1/1) of the monomer to cross-linker, a reaction time over 120 s, a short drying time, (75%) ethanol as the reaction solvent, and low-intensity UV light, largely because these factors together generate optimal three-dimensional networks of cross-links

Illustration of the study workflow.

<p>Illustration of the study workflow.</p

Serum biochemical measurements.

<p>(A) Serum BUN. (B) Serum phosphate. (C) Serum Calcium. (D) Serum 1,25(OH)2D. (E) Serum PTH. (F) Serum FGF23. *: p<0.05 vs Sham; #: p<0.05 vs OVX; $: p<0.05 vs CKD.</p

Histological staining of trabecular bone.

<p>(H&E staining) Buccal lingual cross sections of the furcation area between the mesial root and the distal root of the mandibular first molar. The second row consists of magnified images (400X) of trabecular bone. B: Bone marrow space; C: Cancellous bone.</p

Comparative Analysis of Immune Repertoires between Bactrian Camel's Conventional and Heavy-Chain Antibodies

<div><p>Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (<i>P</i> < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and <i>in silico</i> analysis based on high-throughput NGS data.</p></div

Specific amino acids differences between VH and VHH clones of three camels.

<p>Specific amino acids differences between VH and VHH clones of three camels.</p

Box plots of the mutation rate analysis.

<p><b>a</b> The mutation rates of each sub-region including FR1, CDR1, FR2, CDR2, FR3, and FR4 in three samples before filtering. Mutations are defined as mismatches with the IMGT references on each sample’s sequences. <b>b</b> The mutation rates of each sub-region including FR1, CDR1, FR2, CDR2, FR3, and FR4 in three samples after filtering. <b>c</b> Statistics of specific nucleic acid mutations of the VH and VHH clones before filtering. <b>d</b> Statistics of specific nucleic acid mutations of the VH and VHH clones after filtering. Box plot explanation: upper horizontal line of box, 75th percentile; lower horizontal line of box, 25th percentile; horizontal bar within box, the median of the three samples’ data; upper end of the whisker, maximum of the three samples’ data; lower end of the whisker, minimum of the three samples’ data.</p

Schematic diagram of the CDR3 (AA)-relative analysis and in-frame percent (ORF) statistics.

<p><b>a-c</b> The CDR3-related analysis of the NO.1, NO.2, and NO.3 camels including CDR3 length (AA) and proportion; <b>d</b> The comparison of in-frame percent of antibody variable region genes between VH and VHH clones of three camels.</p

Box plots of the distribution and proportion of Cys codons in VH and VHH clones.

<p>The distribution and proportion of Cys codons (TGC and TGT) in each sub-region including FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in three samples was plotted. The percent equals to the total number of Cys codons divided by the total number of codons in each sub-region. Box plot explanation: upper horizontal line of box, 75th percentile; lower horizontal line of box, 25th percentile; horizontal bar within box, the median of the three samples’ data; upper end of the whisker, maximum of the three samples’ data; lower end of the whisker, minimum of the three samples’ data.</p

Box plots of the analysis of non-classical VHH clones.

<p><b>a</b> The percentage of Trp-clones and Arg-clones (the clones whose first AA of FR4 are Trp and Arg) for classical VHH, non-classical VHH and VH(3) family clones. <b>b</b> Statistics of Cys codons within the Trp-clones and Arg-clones for classical VHH, non-classical VHH, and VH(3) family clones. <b>c</b> The average CDR3 length statistics of Trp-clones and Arg-clones for classical VHH, non-classical VHH and VH(3) family clones. <b>d-f</b> The proportion distribution of CDR3 average length of Trp-clones and Arg-clones. FR4_1st_AA represents the first AA of FR4 sub-region. Classical VHH represents the VHH having four FR2 hallmark amino acids. Non-classical VHH represents the VHH lacking four FR2 hallmark amino acids. VH represents the VH(3) (clan III) family clones. Box plot explanation: upper horizontal line of box, 75th percentile; lower horizontal line of box, 25th percentile; horizontal bar within box, the median of the three samples’ data; upper end of the whisker, maximum of the three samples’ data; lower end of the whisker, minimum of the three samples’ data.</p