Supplemental Material - Associations of Cognitive Impairment with All-Cause and Cardiovascular Mortality Among Individuals with Diabetes: A Prospective Cohort Study

Supplemental Material for Associations of Cognitive Impairment with All-Cause and Cardiovascular Mortality Among Individuals with Diabetes: A Prospective Cohort Study by Yanchang Shang, Shuhui Wang, Chao Wei, and Hengge Xie in Journal of Applied Gerontology</p

van der Waals SWCNT@BN Heterostructures Synthesized from Solution-Processed Chirality-Pure Single-Wall Carbon Nanotubes

Single-wall carbon nanotubes in boron nitride (SWCNT@BN) are one-dimensional van der Waals heterostructures that exhibit intriguing physical and chemical properties. As with their carbon nanotube counterparts, these heterostructures can form from different combinations of chiralities, providing rich structures but also posing a significant synthetic challenge to controlling their structure. Enabled by advances in nanotube chirality sorting, clean removal of the surfactant used for solution processing, and a simple method to fabricate free-standing submonolayer films of chirality pure SWCNTs as templates for the BN growth, we show it is possible to directly grow BN on chirality enriched SWCNTs from solution processing to form van der Waals heterostructures. We further report factors affecting the heterostructure formation, including an accelerated growth rate in the presence of H2, and significantly improved crystallization of the grown BN, with the BN thickness controlled down to one single BN layer, through the presence of a Cu foil in the reactor. Transmission electron microscopy and electron energy-loss spectroscopic mapping confirm the synthesis of SWCNT@BN from the solution purified nanotubes. The photoluminescence peaks of both (7,5)- and (8,4)-SWCNT@BN heterostructures are found to redshift (by ∼10 nm) relative to the bare SWCNTs. Raman scattering suggests that the grown BN shells pose a confinement effect on the SWCNT core

Deletion of C7L and K1L Genes Leads to Significantly Decreased Virulence of Recombinant Vaccinia Virus TianTan

<div><p>The vaccinia virus TianTan (VTT) has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.</p> </div

Comparison of cellular response and humoral response against HIV after immunized with recombinant mutants and VTKgpe.

<p>Comparison of cellular and humoral responses after mice were immunized with VTKgpe, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. Mice were sacrificed one week post-infection, and splenocytes were harvested. (A) The gag specific IFN-γ secreting cells were quantified by ELISPOT assay. (B) The env specific IFN-γ secreting cells were quantified. (C) The gag specific IL-2 secreting cells were quantified. (D) The env specific IL-2 secreting cells were quantified. (E) Serum were harvested and tested for p55-specific antibody levels by ELISA assay. (F) Serum was tested for gp120-specific antibody levels.</p

Virulence analysis in rabbit model.

<p>Cutaneous virulence in rabbits injected intradermally on the dorsal spine with 10-fold dilution of viruses. Ulcer diameters were recorded at day 4 and day 7 post-infection. (A) The size of lesions formed by 10⁷ pfu viruses were shown at day 4 post-infection. (B) The size of lesions formed by 10⁷ pfu viruses were shown at day 7 post-infection. (C) Deletion of the K1L gene resulted in notable decrease in virulence.</p

Virulence analysis in BALB/c mouse.

<p>(A) Groups of 6 BALB/c mice were infected intranasally with 10⁵ pfu of indicated viruses or mock infected with the same volume of PBS. The mice were then monitored daily for their body weights. For each group, the average and standard deviation of the percent of body weight change are shown. (B) 3 week-old mice were inoculated intracranially with 3×10⁴ pfu viruses, and deaths were recorded daily. The virulence in nervous system decreased when K1L gene was deleted. (C) 3 week-old mice were inoculated intracranially with 3×10⁴ pfu viruses. The brains were ground and supernatant used in plaque assays, which revealed reduced virulence in VTT△C7LK1L.</p

Comparison of cellular response and humoral response after immunized with recombinant mutants and VTKgpe.

<p>BALB/c mice were immunized with recombinant mutants and VTKgpe, one week after the final vaccination, mice were sacrificed. (A) Serum was tested for vaccinia specific binding antibody. (B) Serum was tested for vaccinia specific neutralizing antibody. (C) E3 specific IFN-γ secreting cells were not significantly different among viruses. (D) E3 specific IL-2 secreting cells were not significantly different among viruses.</p

Construct of recombinant VTT and western blot analysis.

<p>(A) The deleted genes of vaccinia genome. (B) Construction of VTKgpe△C7L. (C) CEF cells were infected with indicated viruses and harvested at 24h post-infection. Lysates were prepared and analyzed by WB probed with anti-p55 antibody. (D) CEF cells were infected with indicated viruses and harvested at 24h post-infection. Lysates were prepared and analyzed by WB probed with anti-gp120 antibody.</p

Cellular and humoral responses elicited against VTT in mice.

<p>BALB/c mice were immunized with wildtype and deletion mutant VTT, and Mice were sacrificed at week 4 post-infection. (A) Titers of binding antibody elicited by the deletion mutants were notably decreased. (B) Neutralizing antibody titers were defined as the reciprocal of serum dilution that reduced viral plaques by 50%, and the deletion mutants were notably decreased. (C) E3 specific IFN-γ secreting cells immune responses were not significantly different among viruses. (D) E3 specific IL-2 secreting cells immune responses were not significantly different among viruses.</p

Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain

<div><h3>Background</h3><p>Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.</p> <h3>Methods and Findings</h3><p>We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs <em>in vivo</em>.</p> <h3>Conclusions</h3><p>The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.</p> </div