High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Disease-Associated Q159X Mutant Prion Protein Is Sufficient to Cause Fatal Degenerative Disease in Mice.

PRNP Q160X is one of the five dominantly inheritable nonsense mutations causing familial prion diseases. Till now, it remains unclear how this type of nonsense mutations causes familial prion diseases with unique clinical and pathological characteristics. Human prion protein (PrP) Q160X mutation is equivalent to Q159X in mouse PrP, which produces the mutant fragment PrP1-158. Through intracerebroventricular injection of recombinant adeno-associated virus in newborn mice, we successfully overexpressed mouse PrP1-158-FLAG in the central nervous system. Interestingly, high level PrP1-158-FLAG expression in the brain caused death in these mice with an average survival time of 60 ± 9.1 days. Toxicity correlated with levels of PrP1-158-FLAG but was independent of endogenous PrP. Histopathological analyses showed microgliosis and astrogliosis in mouse brains expressing PrP1-158-FLAG and most of PrP1-158-FLAG staining appeared intracellular. Biochemical characterization revealed that the majority of PrP1-158-FLAG were insoluble and a significant part of PrP1-158-FLAG appeared to contain an un-cleaved signal peptide that may contribute to its cytoplasmic localization. Importantly, an ~10-kDa proteinase K-resistant PrP fragment was detected, which was the same as those observed in patients suffering from this type of prion diseases. To our knowledge, this is the first animal study of familial prion disease caused by Q159X that recapitulates key features of human disease. It will be a valuable tool for investigating the pathogenic mechanisms underlying familial prion diseases caused by nonsense mutations.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

Major depressive disease research in BRICS: A bibliometric analysis of publications from 2003 to 2022.

Major depressive disorder (MDD) seriously threatens human health. BRICS, known as an acronym for "Brazil, Russia, India, China, and South Africa," were also actively carrying out researches on MDD. This study aimed to conduct a bibliometric study of research on MDD conducted by the BRICS. By searching in the Web of Science and using the software Vosviewer and Citespace as analysis tools, this study analyzed the cooperation network at the country, institution, author-specific levels, the research hotspots and trends from BRICS between 2003-2022. A total of 10,911 articles were finally included. Our findings showed that researches on MDD from BRICS rapidly increased during the past two decades. China and India have shown explosive growth, while South Africa has the largest average "Usage Count" and "Time Cited". The current cooperation partners of the BRICS were mainly high-income countries and other developing countries with similar cultures, languages, and geographical locations. Institutions in high-income countries served as the main bridges for BRICS cooperation, while at the author level, some core authors in the BRICS countries serve as centers. China showed a flexible model in domestic partnership, but institutions and authors in the other four countries have gathered to cooperate within the group. BRICS research on MDD mainly focused on cognitive science, brain science, epidemiology, and disease mechanisms. The keywords"gut microbiota", "network analysis," "machine learning" and "sleep quality" showed explosive growth and might become research hotspots in the near future. This bibliometric analysis provided a science knowledge graph and references for other researchers.

Barriers to and Facilitators of the Implementation of a Micronutrient Powder Program for Children: A Systematic Review Based on the Consolidated Framework for Implementation Research.

BACKGROUND: As one of the most cost-effective investments for improving child nutrition, micronutrient powder (MNP) has been widely used in many countries to underpin the Sustainable Development Goals, yet challenges remain regarding its implementation on a large scale. However, few studies have explored the factors that facilitate or impede the implementation process using implementation science theories and frameworks. To address this gap, we adopted the Consolidated Framework of Implementation Research (CFIR) and conducted a systematic review of studies on the implementation barriers to and facilitators of MNP interventions. METHOD: Five publication databases, including EMBASE, Medline, PubMed, Web of Science, and Scopus, were searched for studies on the influencing factors of MNP interventions. Based on the CFIR framework, the facilitators and barriers for the MNP program implementation reported in the included studies were extracted and synthesized by five domains: intervention characteristics, outer setting, inner setting, individual characteristics, and process. RESULTS: A total of 50 articles were eligible for synthesis. The majority of the studies were conducted in lower-middle-income countries (52%) through the free delivery model (78%). The inner setting construct was the most prominently reported factor influencing implementation, specifically including available resources (e.g., irregular or insufficient MNP supply), structural characteristics (e.g., public-driven community-based approach), and access to information and knowledge (e.g., lack of training for primary-level workers). The facilitators of the engagement of private sectors, external guidelines, and regular program monitoring were also highlighted. On the contrary, monotonous tastes and occasional side effects impede intervention implementation. Additionally, we found that the inner setting had an interrelation with other contributing factors in the MNP program implementation. CONCLUSION: Our results suggest that MNP program implementation was prominently influenced by the available resources, organizational structure, and knowledge of both providers and users. Mobilizing local MNP suppliers, engaging public-driven free models in conjunction with market-based channels, and strengthening the training for primary-level health workers could facilitate MNP interventions.

Access to DTP-Based Combination Vaccines in Asia-Pacific Countries between 2019 and 2022.

The Asia-Pacific countries are highly diverse in health and economic conditions that may impact vaccine access and uptake. Our study aimed to characterize patterns of health access to DTP-based combination vaccines in 10 countries from 2019 to 2022 using the IQVIA-MIDAS database. The availability, affordability, and accessibility were compared across countries by national health and economic performance indicators using Spearman's rank correlation coefficient. Our findings showed that the three aspects of access to DTP-based vaccines varied substantially in the Asia-Pacific region, with higher levels in countries with better health and economic performance. Affected by the COVID-19 pandemic, vaccine accessibility fluctuates significantly in lower-income countries, with DTP coverage rates falling by more than 14% in the Philippines and Indonesia between 2019 and 2021. For availability and affordability, Singapore and Malaysia from high-income groups were largely affected, which may be related to health expenditure as a percentage of gross domestic product (Coefficient = 0.39, p = 0.03). Our study indicates that greater attention needs to be paid to national health expenditure and routine immunization services to improve vaccine disparities and increase the robustness and resilience of the vaccine supply chain during public health emergencies.