Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses.

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

Students' Perception of a Virtual Dissection Laboratory in Undergraduate Anatomy and Physiology of Speech and Hearing: A Focus Group Study.

Hands-on laboratory experience that allows for manipulation of realistic and relevant materials in course curricula has been shown to improve students' learning, understanding, and critical thinking skills. The purpose of this study was to gain insight into the experiences of students who engaged in laboratory coursework using a virtual dissection (VD) table as part of an undergraduate course in anatomy and physiology of speech and hearing. Undergraduate students enrolled in an anatomy and physiology of speech and hearing course at a single university for the fall 2021 semester consented to participate. Nine students, divided into two focus groups, were encouraged to describe their experiences and perspectives about the VD table and corresponding laboratory assignments. Following verbatim transcription of the data, the authors conducted a thematic analysis. Five themes emerged from the body of data: (1) using the VD table, (2) completing the VD lab assignments, (3) preparation for laboratory sessions, (4) suggested modifications, and (5) enriched learning. Students believed using the VD table aided in a better understanding of course material than traditional methods. Moreover, they surmised that this method of learning, particularly for speech-language pathologists, may be superior to learning through models and cadavers.

Critical and Creative Thinking and Photovoice: Strategies for Strengthening Participation and Inclusion.

Researchers, nonprofit organizations, and others have long used photovoice as a participatory action research method with vulnerable groups to depict, reflect on, and describe their realities, and advocate for change. Paulo Freire, whose scholarship is a foundation of photovoice, encouraged critical thinking in a popular education process to understand "the 'why' of things and facts." Creative thinking, a complementary concept that emerged in the field of education in the 1990s, involves, at its core, development, implementation, and communication of multiple original ideas. We provide a model of critical and creative thinking as an integrated process that generates knowledge as participants complete four key photovoice steps: (1) answer questions with a camera, (2) communicate in the group, (3) observe commonalities, and (4) communicate to power. We argue that each step involves teachable skills and provide practical, low-tech strategies that photovoice facilitators can use to enhance critical and creative thinking by any participant who finds it challenging to complete the four steps. Bringing a critical and creative thinking process to photovoice facilitation grounds the method in its education roots. It can enhance participation and inclusion of any vulnerable group, including people with cognitive and communication disorders due to acquired brain injury, mental illness, or substance use disorder for example. We suggest that use of the suggested strategies will result in an authentic, meaningful process that helps equalize power relationships, respects individuals as experts on their own lives, and increases the potential for data that prompt action.

Coordinated speech therapy, physiotherapy, and pharmaceutical care telehealth for people with Parkinson disease in rural communities: an exploratory, 8-week cohort study for feasibility, safety, and signal of efficacy.

INTRODUCTION: The potential for coordinated, multidisciplinary telehealth to help connect people with Parkinson disease (PD) in rural areas to PD specialists is crucial in optimizing care. Therefore, this study aimed to test the feasibility, safety, and signal of efficacy of a coordinated telehealth program, consisting of speech therapy, physiotherapy, and pharmaceutical care, for people with PD living in some rural US communities. METHODS: Fifteen individuals with PD living in rural Wyoming and Nevada, USA, participated in this single-cohort, 8-week pilot study. Participants were assessed before and after 8 weeks of coordinated, one-on-one telehealth using the following outcomes: (1) feasibility: session attendance and withdrawal rate; (2) safety: adverse events; and (3) signal of efficacy: Communication Effectiveness Survey, acoustic data (intensity, duration, work (intensity times duration)), Parkinson's Fatigue Scale, 30 second Sit-to-Stand test, Parkinson's Disease Questionnaire - 39, Movement Disorder Society Unified Parkinson's Disease Rating Scale - Part III, and medication adherence. RESULTS: Average attendance was greater than 85% for all participants. There were no serious adverse events and only nine minor events during treatment sessions (0.9% of all treatment sessions had a participant report of an adverse event); all nine cases resolved without medical attention. Although 14 of 16 outcomes had effect sizes trending in the direction of improvement, only two were statistically significant using non-parametric analyses: 30 second Sit-to-Stand (pre-test median=11.0 (interquartile range (IQR)=6.0); post-test median=12.0 (IQR=3.0) and acoustic data work (pre-test median=756.0 dB s (IQR=198.4); post-test median=876.3 dB s (IQR=455.5), p<0.05. CONCLUSION: A coordinated, multidisciplinary telehealth program was safe and feasible for people in rural communities who have PD. This telehealth program also yielded a signal of efficacy for most of the outcomes measured in the study.

Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human.

BACKGROUND: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. METHODS: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. RESULTS: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. CONCLUSIONS: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

De novo gene signature identification from single-cell RNA-seq with hierarchical Poisson factorization.

Common approaches to gene signature discovery in single-cell RNA-sequencing (scRNA-seq) depend upon predefined structures like clusters or pseudo-temporal order, require prior normalization, or do not account for the sparsity of single-cell data. We present single-cell hierarchical Poisson factorization (scHPF), a Bayesian factorization method that adapts hierarchical Poisson factorization (Gopalan et al, 2015, Proceedings of the 31st Conference on Uncertainty in Artificial Intelligence, 326) for de novo discovery of both continuous and discrete expression patterns from scRNA-seq. scHPF does not require prior normalization and captures statistical properties of single-cell data better than other methods in benchmark datasets. Applied to scRNA-seq of the core and margin of a high-grade glioma, scHPF uncovers marked differences in the abundance of glioma subpopulations across tumor regions and regionally associated expression biases within glioma subpopulations. scHFP revealed an expression signature that was spatially biased toward the glioma-infiltrated margins and associated with inferior survival in glioblastoma.

Using the OSU TBI-ID method for screening rural, older adults: a mixed methods feasibility study.

Objective: To examine the feasibility of: (1) a brain injury and screening tool (Ohio State University Traumatic Brain Injury Identification Method; OSU TBI-ID) training for rural Area Agency on Aging (AAA) health professionals, and (2) implementation of the screening process.  Research Design: A mixed methods design was utilized to assess several aspects of feasibility. Quantitatively, the researchers examined AAA health professionals' scores on a brain injury misconceptions survey, descriptive statistics about the clients screened, and health professionals' accuracy using the OSU TBI-ID. Qualitative data was collected via focus group interviews with the health professionals.  Methods and Procedures: The researchers conducted brain injury education and screening tool training for AAA health professionals. Subsequently, the health professionals used the OSU TBI-ID to screen their older adult clients for brain injury.  Main Outcomes and Results: AAA health professionals learned the OSU TBI-ID quickly and used the protocol correctly. The screening results established that 15% of the clients screened positive for brain injury; of those with positive screens, 87% did not have a previous brain injury diagnosis.  Conclusions: The current study presents evidence of feasibility for (1) training AAA health professionals, and (2) implementation of the OSU TBI-ID to screen older adult clients.

The diverse vocational experiences of five individuals returning to work after severe brain injury: A qualitative inquiry.

OBJECTIVE: The researchers explored the return-to-work experiences of five adults with severe traumatic brain injuries (TBIs) and those associated with them by performing a qualitative, multiple case study investigation involving in-depth, semi-structured interviews. Selection of this qualitative methodology allowed for personal, individualized accounts of adults with TBI returning to work. Specifically, this methodology promoted accurate representation of the idiosyncratic nature of each participant's experiences. METHODS: The researchers asked individuals to participate based on the diversity of their vocational experiences following TBI. RESULTS: Four of the five participants had returned to their pre-injury jobs. Two were subsequently fired and, at the time of research participation, were unemployed. One participant never returned to paid employment; however, he had held two volunteer positions for several years post-injury. Salient content from interview transcripts allowed for the identification of five to eight themes pertinent to each case. CONCLUSIONS: Interpretation of the themes led to three main conjectures about return-to-work experiences following TBI: (a) job satisfaction may relate more to involvement in productive activities than monetary compensation; (b) adults with TBI can be successful in holding and maintaining positions with high cognitive demands; and

Mobilizing a Statewide Network to Provide Ebola Education and Support.

BACKGROUND: Healthcare providers require the latest information and procedures when a public health emergency arises. During the fall of 2014, when the Ebola virus was first identified in a patient in the United States, education about Ebola virus disease (EVD) and procedures for its identification and control needed widespread and immediate dissemination to healthcare providers. In addition, there was a need to allay fears and reassure the public and providers that a process was in place to manage Ebola should it arrive in Arkansas. The state health department engaged multiple interest groups and provided a variety of educational and management activities. The Arkansas Department of Health and the only academic medical center in the state began offering time-consuming, one-on-one education over the phone, which reached too few providers. A solution was needed to educate many providers across the state in the protocols for identification, isolation, and management of patients with EVD. In response, the Arkansas Department of Health and the University of Arkansas for Medical Sciences leveraged the interactive video and Webinar capabilities of the state's telemedicine network to educate both providers and the public of this public health emergency. MATERIALS AND METHODS: Six interactive video events were staged over 5 days in October 2014. RESULTS: In six events, 82 individual healthcare facilities (67 of which were hospitals) and 378 providers attended via the Webinar option, whereas 323 healthcare professionals received continuing education credits. CONCLUSIONS: A statewide videoconferencing infrastructure can be successfully mobilized to provide timely public health education and communication to healthcare providers and the public in multiple disciplines and practice settings.

Insights on Literacy From Stroke Survivors With Aphasia: A Mixed-Methods Inquiry.

PURPOSE: Individuals with aphasia commonly experience reading and writing difficulties, negatively impacting everyday communication and life participation. Using mixed methods, this study aimed to understand literacy experiences described by individuals with aphasia and explore how their perspectives are related to test performance and other demographic factors. METHOD: Twenty-one stroke survivors with aphasia completed reading and writing testing and shared their perspectives through a close-ended survey and an open-ended interview about literacy abilities and experiences. Quantitative methods were used to compare pre- and poststroke self-ratings and explore associations between self-ratings and demographic factors. Qualitative methods were used to identify themes in the interviews. The data sets were merged to derive mixed-methods results for a more in-depth view of participants' perspectives. RESULTS: Significant decreases in perceived literacy abilities were found; however, there were no differences in literacy importance or enjoyment pre- to poststroke. Reading and writing test scores were correlated with self-rated abilities but not with importance, enjoyment, or frequency of reading and writing. The thematic analysis process identified four main themes: Feelings about literacy, Literacy challenges, Literacy supports, and Literacy goals. CONCLUSIONS: The data indicate that individuals with aphasia highly value reading and writing and are heavily invested, despite recognized challenges, in using and improving these skills. Therefore, assessments and treatments addressing literacy in aphasia are critical, and individuals with aphasia should be invited to share their literacy experiences and goals, allowing for more person-centered clinical resources to be collaboratively constructed. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.25893049.

Identification and Pharmacological Targeting of Treatment-Resistant, Stem-like Breast Cancer Cells for Combination Therapy.

Tumors frequently harbor isogenic yet epigenetically distinct subpopulations of multi-potent cells with high tumor-initiating potential-often called Cancer Stem-Like Cells (CSLCs). These can display preferential resistance to standard-of-care chemotherapy. Single-cell analyses can help elucidate Master Regulator (MR) proteins responsible for governing the transcriptional state of these cells, thus revealing complementary dependencies that may be leveraged via combination therapy. Interrogation of single-cell RNA sequencing profiles from seven metastatic breast cancer patients, using perturbational profiles of clinically relevant drugs, identified drugs predicted to invert the activity of MR proteins governing the transcriptional state of chemoresistant CSLCs, which were then validated by CROP-seq assays. The top drug, the anthelmintic albendazole, depleted this subpopulation in vivo without noticeable cytotoxicity. Moreover, sequential cycles of albendazole and paclitaxel-a commonly used chemotherapeutic -displayed significant synergy in a patient-derived xenograft (PDX) from a TNBC patient, suggesting that network-based approaches can help develop mechanism-based combinatorial therapies targeting complementary subpopulations.

Gluten induces rapid reprogramming of natural memory αß and γδ intraepithelial T cells to induce cytotoxicity in celiac disease.

Celiac disease (CD) is an autoimmune disease in which intestinal inflammation is induced by dietary gluten. The means through which gluten-specific CD4+ T cell activation culminates in intraepithelial T cell (T-IEL)-mediated intestinal damage remain unclear. Here, we performed multiplexed single-cell analysis of intestinal and gluten-induced peripheral blood T cells from patients in different CD states and healthy controls. Untreated, active, and potential CD were associated with an enrichment of activated intestinal T cell populations, including CD4+ follicular T helper (TFH) cells, regulatory T cells (Tregs), and natural CD8+ αß and γδ T-IELs. Natural CD8+ αß and γδ T-IELs expressing activating natural killer cell receptors (NKRs) exhibited a distinct TCR repertoire in CD and persisted in patients on a gluten-free diet without intestinal inflammation. Our data further show that NKR-expressing cytotoxic cells, which appear to mediate intestinal damage in CD, arise from a distinct NKR-expressing memory population of T-IELs. After gluten ingestion, both αß and γδ T cell clones from this memory population of T-IELs circulated systemically along with gluten-specific CD4+ T cells and assumed a cytotoxic and activating NKR-expressing phenotype. Collectively, these findings suggest that cytotoxic T cells in CD are rapidly mobilized in parallel with gluten-specific CD4+ T cells after gluten ingestion.

Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic and targetable tissue states.

Glioblastoma (GBM) diffusely infiltrates the brain and intermingles with non-neoplastic brain cells, including astrocytes, neurons and microglia/myeloid cells. This complex mixture of cell types forms the biological context for therapeutic response and tumor recurrence. We used single-nucleus RNA sequencing and spatial transcriptomics to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional 'tissue-states' defined by cohabitation patterns between specific subpopulations of neoplastic and non-neoplastic brain cells. These tissue-states correlated with radiographic, histopathologic, and prognostic features and were enriched in distinct metabolic pathways. Fatty acid biosynthesis was enriched in the tissue-state defined by the cohabitation of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and macrophages, and was associated with recurrent GBM and shorter survival. Treating acute slices of GBM with a fatty acid synthesis inhibitor depleted the transcriptional signature of this pernicious tissue-state. These findings point to therapies that target interdependencies in the GBM microenvironment.

Considerations for Voice and Communication Training Software for Transgender and Nonbinary People.

Transgender and gender diverse people often experience voice-gender incongruence, which is inversely correlated with health and quality of life. Such incongruence could be reduced with voice and communication training, but expert-administered training is often inaccessible while self-guided training is difficult and potentially risky. Training could alternatively be provided through software (eg, smartphone apps), but such software is at an early stage. This qualitatively driven mixed-methods study thus includes surveys and interviews with 21 transfeminine, transmasculine and nonbinary people to identify general views of voice and communication training software as well as most desirable features of such software. Participants were positive about the general idea and described ways to effectively implement four critical features: feedback, accountability, automated goal setting, and training characteristics other than pitch. They also discussed optional or undesirable features. These findings may inform development of voice and communication training software, thus improving health and quality of life for gender minorities.

Exploring the study skills and accommodations used by college student survivors of traumatic brain injury.

PRIMARY OBJECTIVE: Survivors of severe traumatic brain injury (TBI) recovering sufficiently to attend post-secondary institutions often display learning skills differing from those of traditional students; because of this, many use formal and informal accommodations and learning strategies to support academic performance and social integration. Professionals know little about how accommodations and learning strategies affect educational experiences of college student survivors, how best to apply accommodations and learning strategies, how much survivors embrace them, the extent to which they facilitate college success or how they evolve over time. The purpose of this study was to explore these issues. RESEARCH DESIGN: The researchers collected and analysed qualitative data using a multiple case study design. METHODS AND PROCEDURES: Collected data included interviews, artifacts and field notes from four college student survivors, members of their families, college instructors and personnel from institution's offices for students with disabilities. MAIN OUTCOMES AND RESULTS: Findings suggest challenges, learning strategies, future perspectives and the need for academic accommodations fluctuate and evolve for many years post-injury. Findings also suggest some survivors perceive their learning capabilities more positively than do others associated with them, leading to questions about whether over-accommodation occurs and the potential negative ramifications associated with this practice.

Considering Health Care Needs in a Rural Parkinson Disease Community.

BACKGROUND: People living with Parkinson disease (PD) have multiple health care needs that intensify over time, because the disease is both chronic and degenerative. Past research indicates that issues with mobility, financial constraints, and lack of support networks impede access to health care for people with PD. These challenges are elevated for individuals who live in rural communities due to the lack of local health care professionals and specialists and support resources, and the need to travel to see providers/specialists. The research objective was to have PD community stakeholders identify health care barriers and resources as well as possibilities for improved health care in a rural state. METHODS: Focus groups were conducted in the context of a community-based participatory research (CBPR) approach. Focus group data collection helped create comfort and parity in the discussion, while a CBPR approach allows for authenticity of the findings because members of the community in question are involved as researchers. The responses were recorded and transcribed verbatim. Coding and organizing of themes was completed manually and using NVIVO 10 software. RESULTS: Qualitative analysis revealed three main themes, PD issues, access issues, and stigma. These themes described disease-related stigma and concerns about disease progression and treatment, as well as challenges in accessing information, providers, and support by the PD stakeholders. The study results provided insight into the needs of people living with PD in rural communities. CONCLUSIONS: Rural PD stakeholders proposed the use of technology (e.g., telehealth) to provide support to improve health care for people with PD.

Describing the Composition of Individualized Education Plans for Students With Traumatic Brain Injury.

Purpose The purpose of this study was to explore and describe the features of Individualized Education Plans (IEPs) for a cohort of students with traumatic brain injury (TBI) to help elucidate current special education practices for students with TBI. Method We obtained permission from administrators of a local school district of 41,000 students in a Midwestern state to review de-identified IEP records of students verified with TBI. We examined demographic information (i.e., cause and age at time of injury), IEP services and intensity, IEP goal categories, and previous verification status. Results Descriptive results support that intervention services were more intense for students with TBI with greater lengths of time postinjury. Target behaviors within goals were more often related to math and reading than to the cognitive processes that govern these skills, such as attention, memory, and executive functioning. Finally, more than a third of our sample had been verified with a disability and were receiving special education services via an IEP prior to their TBI. Conclusions This work represents an important first step in understanding the special education services for students with TBI. Future research should explore interventions that are ecologically valid for school-based settings and are developed to address the idiosyncratic deficits of students with TBI, particularly interventions that focus on the underlying cognitive processes experienced by these students.

Single-Cell Transcriptional Profiling of the Intestinal Epithelium.

Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmental perturbations. scRNA-seq is particularly useful for characterizing the intestinal epithelium because it is a dynamic, rapidly self-renewing tissue comprised of more than a dozen specialized cell types. Here we discuss the fundamentals of single-cell transcriptomics of the murine small intestinal epithelium. We review the principles of proper experimental design and provide methods for the dissociation of the small intestinal epithelium into single cells followed by fluorescence-activated cell sorting (FACS) and for scRNA-seq using the 10× Genomics Chromium platform.

Speech-Language Pathologists' Comfort Providing Intervention to Children With Traumatic Brain Injury: Results From a National Survey.

Purpose This exploratory study examined speech-language pathologists' (SLPs) clinical experience and work environment characteristics impacting comfort with providing intervention to children with traumatic brain injury (TBI). Method This study included 162 SLPs who responded to a national survey about their comfort providing intervention to children with TBI, clinical experience (i.e., years of experience treating children with TBI, TBI preprofessional training and professional development, and licensure/credentialing), and work environment (i.e., work setting, caseload size, geographic location). Results Findings from latent class analysis revealed 3 distinct groups of SLPs based on their comfort with providing services to children with TBI: those with low comfort, moderate comfort, and high comfort. Further analyses revealed statistically significant differences across the 3 groups in the areas of years of experience treating children with TBI, professional development, work setting, TBI caseload size, and geographic location. Conclusions Our findings reveal that most SLPs feel comfortable providing intervention to children with TBI; however, differences in characteristics across groups suggest that specific steps can be taken to ensure increased comfort for all SLPs working with this population. Practicing SLPs may increase their level of comfort through professional development and hands-on, mentored experience with TBI. Efforts such as these may influence the quality of service provision and expand the population of SLPs who feel comfortable treating children with TBI. Future research is needed to further examine how comfort and SLP characteristics directly impact the quality of speech and language intervention and long-term outcomes of children with TBI.

Single cell RNA-Seq reveals pre-cDCs fate determined by transcription factor combinatorial dose.

BACKGROUND: Classic dendritic cells (cDCs) play a central role in the immune system by processing and presenting antigens to activate T cells, and consist of two major subsets: CD141+ cDC (cDC1) and CD1c+ cDC (cDC2). A population of migratory precursor cells, the pre-cDCs, is the immediate precursors to both cDC subsets. Previous studies showed that there were two pre-committed pre-cDC subpopulations. However, the key molecular drivers of pre-commitment in human pre-cDCs were not investigated. RESULTS: To identify the key molecular drivers for pre-commitment in human pre-cDCs, we performed single cell RNA sequencing (RNA-Seq) of two cDC subsets and pre-cDCs, and bulk RNA-Seq of pre-cDCs and cDCs from human peripheral blood. We found that pre-DC subpopulations cannot be separated by either variable genes within pre-cDCs or differentially expressed genes between cDC1 and cDC2. In contrast, they were separated by 16 transcription factors that are themselves differentially expressed or have regulated targets enriched in the differentially expressed genes between bulk cDC1 and cDC2, with one subpopulation close to cDC1 and the other close to cDC2. More importantly, these two pre-cDC sub-populations are correlated with ratio of IRF8 to IRF4 expression level more than their individual expression level. We also verified these findings using three recently published datasets. CONCLUSIONS: In this study, we demonstrate that single cell transcriptome profiling can reveal pre-cDCs differentiation map, and our results suggest the concept that combinatorial dose of transcription factors determines cell differentiation fate.