AviTrap: A novel solution to achieve complete biotinylation.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

i-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists.

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.

Khmer Girls in Action and healing justice: Expanding understandings of anti-Asian racism and public health solutions.

This community case study highlights how Khmer Girls in Action (KGA), a Southeast Asian young women-led organizing group in Long Beach, California, enacts healing justice. Healing justice is a framework for both transforming structures at the crux of health inequities and healing emotional, spiritual, and psychological wounds inflicted by structural violence. KGA also anchors the cross-racial and intersectional Invest in Youth (IIY-LB) coalition. IIY-LB youth leaders have successfully fought to increase the city's investments in the social determinants of health, especially young people's well-being. Meanwhile, the coalition has critiqued over-investments in criminalization and policing as devastating Black, Brown, queer, low-income, immigrant, and refugee youth and communities. This case study highlights how KGA's work expands understandings of both anti-Asian racism and public health solutions in the following ways: First, KGA cultivates youth leaders' critical analyses to define root causes of health inequities impacting Southeast Asian refugees as rooted in imperialism, disinvestment, and increased criminalization. Furthermore, youth leaders come to understand how their communities' struggles and liberation necessitate intersectional and cross-racial coalitions. Second, youth leaders forge public health solutions that involve divesting from criminalization and institutionalizing an Office of Youth Development, as co-created with young people. Third, KGA and other IIY-LB organizations cultivate youth's leadership skills and community's political power to move hearts and minds of decision-makers and community members. For example, youth leaders have passed a ballot measure funding youth, climate, and health programs in addition to the city-based Office of Youth Development. Fourth, KGA engages in a wide range of "inward" healing practices to salve wounds caused by intergenerational trauma. This case study contributes to Asian American health equity by highlighting the specific importance of organizing, while illuminating abolitionist perspectives on public health solutions- both of which are under-discussed in discourse about anti-Asian racism. KGA's work thus illustrates the importance of centering critical analyses and leadership of communities most impacted by structural violence in forging transformative public health solutions to anti-Asian racism.

Glycaemic control mediates the relationships of employment status and self-stigma with self-care behaviours in young adults with type 2 diabetes.

AIMS AND OBJECTIVES: To investigate the relationships of sociodemographic factors, self-stigma, glycaemic control (measured by glycated haemoglobin (A1C)) and self-care behaviours in young adults with type 2 diabetes. BACKGROUND: Young adults aged 25-44 years are in their most productive period. Once diagnosed with diabetes, this population tends to experience poor glycaemic control and perform poorly in self-care activities. Such patterns may raise perceptions of self-stigma and further decrease motivations to engage in self-care behaviours in patients with diabetes. DESIGN: A cross-sectional, correlational research design. METHODS: The STROBE guidelines for cross-sectional studies were followed. A convenience sample of 115 participants was recruited from a medical centre in southern Taiwan. Instruments included the Self-Stigma Scale-Chinese version and the Diabetes Self-Care Behaviours Scale. Data were analysed using a three-step hierarchical regression analysis and the Sobel test. RESULTS: The average age of the participants was 36.7 years. Marital status, employment status, self-stigma and A1C were significantly associated with self-care behaviours, and these four variables explained 43.6% of the variance in self-care behaviours. However, A1C (ß = -.58, p < .001) was found to be the only determinant of self-care behaviours in the last regression model. The Sobel test showed that A1C had mediating effects on self-stigma and self-care behaviours as well as employment status and self-care behaviours. CONCLUSION: This study supports the interactive relationship among self-stigma, employment status, glycaemic control and self-care behaviours in young adults with type 2 diabetes. Strategies aimed at optimising glycaemic control can help reduce the effects of self-stigma perceptions and employment status on the self-care behaviours of such patients. RELEVANCE TO CLINICAL PRACTICE: More effective educational programmes should be designed to improve glycaemic control, lower the effects of employment and decrease perceptions of self-stigma to further motivate young adults to engage in better diabetes self-care behaviours.

Cordycepin inhibits the proliferation of malignant peripheral nerve sheath tumor cells through the p53/Sp1/tubulin pathway.

Neurofibromatosis type 1 (NF1) is one of the most common hereditary neurocutaneous disorders. In addition to skin pigmentation and cutaneous neurofibroma, some patients developed the plexiform neurofibroma since birth. Plexiform neurofibroma has abundant Schwann cells, fibroblasts, mast cells, blood vessels, and connective tissues, which increases the risk of developing a malignant peripheral nerve sheath tumor (MPNST). MPNST is a highly invasive cancer with no effective therapeutic agent. Cordycepin or 3'-deoxyadenosine is an extract from cordyceps militaris, which has been reported as an anti-inflammation and anti-tumor agent. Herein, we evaluated cordycepin's anti-proliferative effect on MPNST cell lines both in vitro and in vivo. Cordycepin inhibited the MPNST cell growth with an arrest of cell cycle at G2/M and S phases. The administration of naringin and pentostatin, inhibitors for adenosine deaminase (ADA), enzyme responsible for cordycepin degradation, did not show a synergistic effect in MPNST cells treated with cordycepin. However, the combined treatment enhanced the decrease of tumors in xenograft mouse model. Immunoblotting showed a decreased level of p53 protein in all MPNST cell lines, but S462TY cells. After cordycepin treatment, the levels of ERK, survivin, pAKT, and Sp1 proteins also decreased. The level of tubulin, but not actin or GAPDH, decreased in a dose-dependent manner. The microtubule network which is composed of tubulins was markedly decomposed in those treated MPNST cells. To elucidate the epigenetic control of transcription, ChIP-qPCR assay of the Sp1 and tubulin promoter regions revealed decreased Sp1 binding. The incorporation of 3'-doexyadenosine is detrimental for the process of poly(A) tail elongation. The poly(A) tail length assay showed the tail length in Sp1 and tubulin transcripts decreased in the treated cells. Nevertheless, the administration of SP1 protein to the treated cells could not rescue them completely. Furthermore, the p53-knocked-down cells (S462TY) where the expression of both p53 and Sp1 was suppressed, were vulnerable to cordycepin. The p53 protein could ameliorate the effect. In summary, cordycepin is effective to inhibit the growth of MPNST, probably through the pathway of p53/Sp1/tubulin.

Nanolipoprotein particles for co-delivery of cystine-knot peptides and Fab-based therapeutics.

Nanolipoprotein particles (NLPs) have been evaluated as an in vivo delivery vehicle for a variety of molecules of therapeutic interest. However, delivery of peptide-like drugs in combination with therapeutic Fabs has not yet been evaluated. In this study, we describe the development and characterization of cystine-knot peptide (CKP)-containing NLPs and Fab-CKP-NLP conjugates. CKPs were incorporated into NLPs using a self-assembly strategy. The trypsin inhibitor EETI-II, a model CKP, was produced with a C16 fatty acyl chain to enable incorporation of the CKP into the lipid bilayer core during NLP assembly. The CKP-NLP retained trypsin inhibitory function although the overall activity was reduced by ∼5 fold compared to free CKP, which was presumably due to steric hindrance. The NLP platform was also shown to accommodate up to ∼60 CKP molecules. Moreover, the stability of the CKP-NLP was comparable to the NLP control, displaying a relatively short half-life (∼1 h) in 50% serum at 37 °C. Therapeutic Fabs were also loaded onto the CKP-NLP by introducing thiol-reactive lipids that would undergo a covalent reaction with the Fab. Using this strategy, Fab loading could be reliably controlled from 1-50 Fabs per CKP-NLP and was found to be independent of CKP density. Surprisingly, Fab incorporation into CKP-NLPs led to a substantial improvement in NLP stability (half-life > 24 h) at 37 °C; also, there was no reduction in CKP activity in the Fab-CKP-NLP conjugates compared to CKP-NLPs. Altogether, our data demonstrate the potential of NLPs as a promising platform for the targeted or multidrug delivery of peptide-based drug candidates in combination with Fabs.

Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics.

Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.

Corrigendum to "Prevalence of Dental Caries in 5- and 6-Year-Old Myanmar Children".

[This corrects the article DOI: 10.1155/2019/5948379.].

Prevalence of Dental Caries in 5- and 6-Year-Old Myanmar Children.

There are no national data available of the oral health in Myanmar. In this study, we examined dental caries status of 187 school children located in the suburban area of Naypyidaw, capital of Myanmar, at the age of five and six and analyzed by the individual level and tooth level. Maxillary D and B were sensitive for dental caries almost at the same level. They were less sensitive than maxillary A. Mandibular A and B were tolerant for dental caries. Prevalence of dental caries in Myanmar children was still high. By applying item response theory and multilevel modeling, tooth level analysis can be implemented to confirm the tendency for sensitivity or tolerance for dental caries by the tooth level.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.