Data_Sheet_1_Exploring functional metabolites and proteomics biomarkers in late-preterm and natural-born pigs.docx

IntroductionPigs are often used to study the intestinal development of newborns, particularly as preterm pig models that mimic the intestinal growth of human preterm infants. Neonatology’s study of delivery mode’s impact on neonatal development is crucial.MethodsWe established 14 newborn pigs delivered via cesarean sections (C-section, at 113 days of gestational age, CS group) and 8 naturally born pigs were used as controls (at 114 days of gestational age, NF group). The impact of two alternative delivery procedures (C-section and natural birth) on the levels of short-chain fatty acids (SCFAs) and organic acids in the hepatic and intestines of newborn pigs were compared using metabolomics. The underlying molecular pathways are examined at the “protein-metabolite” level by integrating proteomic data.ResultsThe findings demonstrated that the mode of delivery changed the metabolism of SCFAs in newborn pigs, perhaps by affecting the physiology levels of cyclic intermediates such as lactate and malate in the pyruvate metabolic pathway. Additionally, by participating in the fatty acid metabolism pathway, two distinct proteins (FASN and HSD17B4) may impact the physiological concentration of these tiny metabolites.DiscussionIn conclusion, this study provided reliable animal model data for understanding the physiological SCFA metabolic information and its affecting mechanism of large-gestational age preterm infants.</p

The growth of E56-old PESPs after implantation.

<p>(a) The PESPs were grafted on the wound and protected by the adult white BAMA pini-pig skin. (b and c) The wound was healed first by host mouse epidermis 2 and 3 weeks postimplantation. (d and e) After 4 weeks of implantation, black color became visible. Finally, the black hair grew out. (f) The dermal side of the black after 6-week implantation.</p

The appearance time of black color and black hair after PESPs posttransplantation.

<p>The appearance time of black color and black hair after PESPs posttransplantation.</p

The histology of E56-old PESPs after 12-week implantation.

<p>(a) The neoregenerative tissue containing melanin was stained by means of Fontana (indicated by arrow). The epidermal and dermal rete ridges were shown by arrowhead. (b) The sebaceous gland was indicated by arrowhead. (c) The sweat gland ducts were indicated by arrow.</p

Growth potential of PESPs from different gestational ages posttransplantation.

<p>E42-old PESPs possessed the maximal growth potential. There were significant difference between E42 and E35 (p = 0.001), E42 and E70 (p = 0.001), E42 and E91 (p = 0.001). The number of animals at each time point was 12. There was no significant difference between E42 and E56 (p = 0.21).</p

The teratoma-like structure of E42-old PESPs after 6-week implantation.

<p>Cartilage tissue and cutaneous tissue were found in neoregenerative tissues (HE, ×200 for larger photo, ×400 for smaller photo).</p

Developmental histology of E35, E42, E56, E72 and E91.

<p>(a) The histological image of E35. (b) The naked image of E42. (c) The histological image of E42. (d) The histological image of E56. Hair follicle-like structure is indicated by arrow. (e) The histological image of E72. The sebaceous gland is indicated by arrow head. (f) The histological image of E91. The duct of sweat gland is indicated by arrow head.</p

The immunohistological staining of neoregenerative tissues after 6–12 week implantation of E56 PESPs.

<p>(a) Cytokeratin MNF116 positive cells were located in epithenium (12-week postimplantation). (b) Cytokeratin MNF116 positive cells were located in hair follicle sheet (6-week postimplantation). (c) Vimentin 9 positive cells were located in the dermis around hair follicles (6-week postimplantation). (d) Vimentin 9 positive cells were located in the deep dermis tissue under the host tissue (12-week postimplantation).</p

Image_1_Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.tif

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.</p

Table_1_Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.xlsx

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.</p