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Background Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. Methods AIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. Results We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. Conclusions We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

The COVID-pandemic has required changes to healthcare delivery and has been a stressful time for people with diabetes mellitus (DM) . Previous literature suggests that virtual health appointments for diabetes care can be effective and result in high patient satisfaction. However, it is unclear if patients with DM are satisfied with the widespread adoption of virtual care during the pandemic. The aim of this study was to evaluate the impact of virtual care during the pandemic on patient satisfaction in patients with type 1 and type 2 DM. The validated Patient Satisfaction Questionnaire (PSQ-III) was completed by 197 patients who had an in-person appointment in the six months before March 18, 2020 (pre-COVID) and a subsequent virtual appointment within six months after that date. For each form of healthcare delivery (i.e. in-person and virtual) , the satisfaction in six aspects of care was measured: general satisfaction, technical quality, interpersonal manner, communication, time spent with doctor, and accessibility of care. Both type 2 DM and age >55 years were associated with decreased general satisfaction with virtual care compared to in-person care. Both type 2 DM and female sex were associated with decreased technical quality of care. Accessibility of care decreased significantly in the overall study group. No significant differences in patient satisfaction were observed in the type 1 DM group. Despite our findings, 74% of respondents answered they would consider virtual health appointments in the future. Our data suggests that many patients welcome virtual health as an addition to their medical care. However, virtual health does not seem to deliver the same level of patient satisfaction as in-person appointments over the longer term. Future research on how fatigue with pandemic restrictions has affected patient satisfaction is needed to evaluate whether virtual health appointments are a viable option for long term diabetes care. Disclosure C.Chang: None. A.Dissanayake: None. M.Pawlowska: Advisory Panel; Novo Nordisk, Other Relationship; Medtronic. B.Schroeder: Advisory Panel; Novartis Canada, Novartis Canada, Novartis Canada, Novo Nordisk Canada Inc., Novo Nordisk Canada Inc., Novo Nordisk Canada Inc. J.Mackenzie-feder: Advisory Panel; Recordati S.p.A. A.White: Advisory Panel; AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, HLS Therapeutics Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk Canada Inc.

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for central nervous system demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination promoting compounds, we generated an advanced, genome engineered, human pluripotent stem cell (hPSC) line that consist of three reporters (identification-and-purification tag, GFP, and secreted NanoLuc) driven by the endogenous PDGFRα, PLP1 and MBP genes, respectively. Based upon this line, we established a high-throughput drug screening platform and performed a small molecule screen with 2500 bioactive molecules. In addition to a number of previously known pathways, our screening effort identified new pathways whose inhibition enhance oligodendrocyte maturation and myelination. Although further genetic and molecular validation is required, the identified inhibitors could potentially be repurposed to develop remyelination therapy for MS and other demyelinating disorders.

Indeed, intelligent model is a sub-domain in computer science that its purposes utilized in numerous pieces of reality. Particularly in case of automotive industry, to select the most appropriate personal vehicle is challenging for the customers. The challenge was taken as a main problem of this study. In this research, the reasonableness is characterized by the customer parameters which portray the customer’s identity. By utilizing the mix technique for fuzzy-logic and Euclidean distance calculation, the customer’s identities are fitted into the personal-vehicle parameters. At long last, the constructed model for choosing personal vehicle plays several customer’s parameters; e.g., age, gender, education, income and job. The model designed thru utilizing the object-oriented methodology. The result of simulation of 66 purchasers and 44 conceivable vehicles are able to propose the most reasonable vehicle for every purchaser. As an extended version, the model successfully delivers the completed scheme due to added parameters and method. It is a truthful contribution provided by this research.

Data on the health and social determinants for Native Hawaiians and Pacific Islanders (NHPIs) in the United States are hidden, because data are often not collected or are reported in aggregate with other racial/ethnic groups despite decades of calls to disaggregate NHPI data. As a form of structural racism, data omissions contribute to systemic problems such as inability to advocate, lack of resources, and limitations on political power. The authors conducted a data audit to determine how US federal agencies are collecting and reporting disaggregated NHPI data. Using the COVID-19 pandemic as a case study, they reviewed how states are reporting NHPI cases and deaths. They then used California's neighborhood equity metric—the California Healthy Places Index (HPI)—to calculate the extent of NHPI underrepresentation in communities targeted for COVID-19 resources in that state. Their analysis shows that while collection and reporting of NHPI data nationally has improved, federal data gaps remain. States are vastly underreporting: more than half of states are not reporting NHPI COVID-19 case and death data. The HPI, used to inform political decisions about allocation of resources to combat COVID-19 in at-risk neighborhoods, underrepresents NHPIs. The authors make recommendations for improving NHPI data equity to achieve health equity and social justice.

As of March 2021, Native Hawaiians and Pacific Islanders (NHPIs) in the United States have lost more than 800 lives to COVID-19—the highest per capita death rate in 18 of 20 US states reporting NHPI deaths. However, NHPI risks are overlooked in policy discussions. We discuss the NHPI COVID-19 Data Policy Lab and dashboard, featuring the disproportionate COVID-19 mortality burden for NHPIs. The Lab democratized NHPI data, developed community infrastructure and resources, and informed testing site and outreach policies related to health equity.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. We have previously developed a biostimulatory nanofiber-hydrogel composite (NHC) through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofibre fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, we improved injectability of the pre-formed NHC by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodelling. The mfNHC showed similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiated an elevated level of host macrophage infiltration, more pro-regenerative polarization and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response was detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodelling.

Mesenchymal stem cells (MSCs) have been widely studied as a versatile cell source for tissue regeneration and remodeling due to their potent bioactivity, which includes modulation of inflammation response, macrophage polarization toward proregenerative lineage, promotion of angiogenesis, and reduction in fibrosis. This review focuses on profiling the effects of paracrine signals of MSCs, commonly referred to as the secretome, and highlighting the various engineering approaches to tune the MSC secretome. Recent advances in biomaterials‐based therapeutic strategies for delivery of MSCs and MSC‐derived secretome in the form of extracellular vesicles are discussed, along with their advantages and challenges., This review discusses various engineering approaches to tune the effects of paracrine signals secreted by mesenchymal stem cells (MSCs), commonly referred to as the secretome. Recent advances in biomaterials‐based therapeutic strategies for delivery of MSCs and MSC‐derived secretome in the form of extracellular vesicles are discussed along with their advantages and challenges.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, we performed single-cell-transcriptomic-analysis of developing human oligodendrocyte-precursor-cells (hOPCs). We engineered knock-in hESC-reporter lines in which an Identification-and-Purification tag is expressed under control of the endogenous, OPC-specific,PDGFRαpromoter, and performed time-course single-cell-RNA-sequencing of purified hOPCs. Our analysis uncovered marked transcriptional heterogeneity of PDGFRα+ hOPCs and identified regulatory genes and networks that control their differentiation and myelination competence. Pseudotime trajectory analysis revealed two distinct trajectories for the development of oligodendrocytes vs astrocytes from hOPCs. We also identified novel transcription factors and other genes that developing hOPCs potentially use to choose between oligodendrocyte vs astrocyte lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of those pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.

一對一數位學習觀念已逐漸被社會所接受，但對實際教學現場的影響仍十分有限；這是 因為將數位學習落實於教室，進而形成「數位學校」仍遭遇許多困難。研究團隊歷經十多年 發展，自 2009 年，以長期採納實踐的模式為主，探索並驗證學生、教師與家長投入數位學習 的意願與改變，逐步發展支援親師生的數位學習模式與平台，目標分別為：學生利用各式的 數位資源主動投入自主學習或同儕互動；教師發展與落實數位教學的能力與課程；家長善用 數位設備與資源來幫助學生。目前導入至數位學校已 6 年，發展全國第一所由家長為自己孩 子採購電腦的 BYOD 模式，並達成學生學科成就明顯提升、教師學與教的典範轉移與家長態 度從忐忑到信任等成果。後續將基於此成果與模式持續推廣與擴散。 Although the notion of one-to-one learning technology is a recent development, its impact on schools has thus far been limited. To develop a feasible model for digital schools based on one-to-one learning technology, the present study employed an adoption-based research methodology to investigate how students, teachers, and parents have engaged with digital learning platforms since 2009. Through the process of understanding their willingness to use digital learning platforms as well as reasons for changing for better learning and teaching, the researchers gradually developed digital learning models and corresponding platforms. The study objectives are described as follows: (1) to facilitate students’ active participation in self-directed learning and collaborative learning by using various digital resources; (2) to assist teachers in developing their capabilities for adopting digital teaching in classrooms; and (3) to assist parents in helping their children through appropriate use of digital devices. Based on these objectives, the present study established the first digital school in Taiwan using a bring-your-own-device model, where parents prepare personal tablet computers for their children. The results show that learning outcomes significantly improved among the students. In addition, the teachers transformed the paradigm of learning and teaching in classrooms, and the parents developed trust in digital learning. Based on these outcomes, the model designed in this study could be used in schools in the future.

In Indonesia, the issue of waste is definitely challenging. This problem demands an exclusive and prudent treatment to implement; as a destructive consequence practically generated from incorrect strategy implementation in treating is wide-ranging, particularly for human’s life and health and also for environment. This paper offers a generic smart ecological decision support model (ecoDSM) for treating medical waste. The mathematical model based on fuzzy logic conception that combined with the other heuristic optimization method are functioned to measure and justify six alternatives of treating strategy decision. Based on empirical data of two types of medical waste (i.e. sharp and infectious) coming from private hospital; the model’s simulation result shows that the "mechanical processing and chemical disinfectant" treatment strategy is the most objective decision should be implemented by the hospital.

This essay examines the importance of disaggregating Native Hawaiian and Pacific Islander data, issues currently faced by NHPI communities, and where NHPI communities could be in 2040. Projected demographic trends may exacerbate challenges faced by NHPIs in areas such as health, education, income, incarceration, housing, and immigration. The impact of climate change, technological innovations, and the United States’ shift towards a majority-minority status on NHPI communities are also analyzed. Three recommendations for improving the position of NHPIs in 2040 are provided: (1) Address the needs of an increasingly diverse NHPI community; (2) develop community capacity for civic engagement; and (3) invest in leadership development and NHPI youth.

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

T cell therapies require the removal and culture of T cells ex vivo to expand several thousand-fold. However, these cells often lose the phenotype and cytotoxic functionality for mediating effective therapeutic responses. The extracellular matrix (ECM) has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies. Here, a hyaluronic acid (HA)-based hydrogel is engineered to present the two stimulatory signals required for T-cell activation—termed an artificial T-cell stimulating matrix (aTM). It is found that biophysical properties of the aTM—stimulatory ligand density, stiffness, and ECM proteins—potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM results in a rapid and robust expansion of rare, antigen-specific CD8+ T cells. Adoptive transfer of these tumor-specific cells significantly suppresses tumor growth and improves animal survival compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM-mimetic materials for therapeutic immune stimulation in the future.

DNA damage-activated nuclease identified Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive. Science , this issue p. 82 ; see also p. 36

Identification and validation of a drug discovery target is a prominent step in drug development. In the post-genomic era it is possible to reevaluate the association of a gene with a specific biological function to see if a homologous gene can subsume this role. This concept has special relevance to drug discovery in human infectious diseases, like malaria. A trophozoite cysteine protease (falcipain-1) from the papain family, thought to be responsible for the degradation of erythrocyte hemoglobin, has been considered a promising target for drug discovery efforts owing to the antimalarial activity of peptide based covalent cysteine protease inhibitors. This led to the development of non-peptidic non-covalent inhibitors of falcipain-1 and their characterization as antimalarials. It is now clear from sequencing efforts that the malaria genome contains more than one cysteine protease and that falcipain-1 is not the most important contributor to hemoglobin degradation. Rather, falcipain-2 and falcipain-3 appear to account for the majority of cysteine hemoglobinase activity in the plasmodium trophozoite. We have modeled the falcipain-2 cysteine protease from one of the major human malaria species, Plasmodium falciparum and compared it to our original work on falcipain-1. As with falcipain-1, computational screening of the falcipain-2 active site was conducted using DOCK. Using structural superpositions within the protease family and evolutionary analysis of substrate specificity sites, we focused on the commonalities and the protein specific features to direct our drug discovery effort. Since 1993, the size of the Available Chemicals Directory had increased from 55313 to 195419 unique chemical structures. For falcipain-2, eight inhibitors were identified with IC50’s against the enzyme between 1 and 7 µM. Application of three of these inhibitors to infected erythrocytes cured malaria in culture, but parasite death did not correlate with food vacuole abnormalities associated with the activity of mechanistic inhibitors of cysteine proteases like the epoxide E64. Using plasmodial falcipain proteases, we show how a protein family perspective can influence target discovery and inhibitor design. We suspect that parallel drug discovery programs where a family of targets is considered, rather than serial programs built on a single therapeutic focus, will become the dominant industrial paradigm. Economies of scale in assay development and in compound synthesis are expected owing to the functional and structural features of individual family members. One of the remaining challenges in post-genomic drug discovery is that inhibitors of one target are likely to show some activity against other family members. This lack of specificity may lead to difficulties in functional assignments and target validation as well as a complex side effect profile.

Satellite cells are the chief contributor to skeletal muscle growth and regeneration. The study of mouse satellite cells has accelerated in recent years due to technical advancements in the isolation of these cells. The study of human satellite cells has lagged and thus little is known about how the biology of mouse and human satellite cells compare. We developed a flow cytometry-based method to prospectively isolate human skeletal muscle progenitors from the satellite cell pool using positive and negative selection markers. Results show that this pool is enriched in PAX7 expressing cells that possess robust myogenic potential including the ability to give rise to de novo muscle in vivo. We compared mouse and human satellite cells in culture and identify differences in the elaboration of the myogenic genetic program and in the sensitivity of the cells to cytokine stimulation. These results indicate that not all mechanisms regulating mouse satellite cell activation are conserved in human satellite cells and that such differences may impact the clinical translation of therapeutics validated in mouse models. Thus, the findings of this study are relevant to developing therapies to combat muscle disease.

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed “mechano-growth factor” (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.