Cell Factors Research Articles

Identification of cambium stem cell factors and their positioning mechanism

Wood constitutes the largest reservoir of terrestrial biomass. Composed of xylem, it arises from one side of the vascular cambium, a bifacial stem cell niche that also produces phloem on the opposing side. It is currently unknown which molecular factors endow cambium stem cell identity. Here we show that TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) ligand–activated PHLOEM INTERCALATED WITH XYLEM (PXY) receptors promote the expression of CAMBIUM-EXPRESSED AINTEGUMENTA-LIKE (CAIL) transcription factors to define cambium stem cell identity in the Arabidopsis root. By sequestrating the phloem-originated TDIF, xylem-expressed PXY confines the TDIF signaling front, resulting in the activation of CAIL expression and stem cell identity in only a narrow domain. Our findings show how signals emanating from cells on opposing sides ensure robust yet dynamically adjustable positioning of a bifacial stem cell layer.

Predicting radiation pneumonitis in lung cancer using machine learning and multimodal features: a systematic review and meta-analysis of diagnostic accuracy.

To evaluate the diagnostic accuracy of machine learning models incorporating multimodal features for predicting radiation pneumonitis in lung cancer through a systematic review and meta-analysis. Relevant studies were identified through a systematic search of PubMed, Web of Science, Embase, and the Cochrane Library from October 2003 to December 2023. Additional studies were located by reviewing bibliographies and relevant websites. Two independent researchers screened titles, abstracts, and full-text articles according to predefined inclusion and exclusion criteria. Data extraction was performed using standardized forms, and study quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The primary outcomes, including combined sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC), were calculated using STATA MP-64 software(Stata Corporation LLC, College Station, USA) with a random-effects model. Meta-analysis was conducted to synthesize diagnostic accuracy measures, and analyses of heterogeneity and publication bias were performed. A total of 1,406 patients with primary lung cancer were included in this systematic review, drawing data from 9 studies. The pooled analysis revealed a sensitivity of 0.74 [0.58-0.85] and a specificity of 0.91 [0.87-0.95] for machine learning models in diagnosing radiation pneumonitis. The positive likelihood ratio (PLR) was 8.69 [5.21-14.50], the negative likelihood ratio (NLR) was 0.28 [0.16-0.49], and the diagnostic odds ratio (DOR) was 30.73 [11.96-78.97]. The area under the curve (AUC) was 0.93 [0.90-0.95], indicating excellent diagnostic performance. Meta-regression analysis identified that the number of machine learning models, year of publication, and study design contributed to heterogeneity among studies. No evidence of publication bias was found. Overall, machine learning models incorporating multimodal characteristics demonstrated 75% accuracy in predicting moderate to severe radiation pneumonitis. In conclusion, by integrating the current machine learning (ML) algorithm's ability in big data mining, a predictive model can be constructed by combining multi-modal features such as genetics, imaging, and cell factors. By selecting multiple machine learning algorithm frameworks and competing for the best combination model based on research goals, the reliability and accuracy of the radiation pneumonitis prediction model can be greatly improved. PROSPERO (CRD42024497599).

Recent Advances of Exosomes Derived from Skeletal Muscle and Crosstalk with Other Tissues.

Skeletal muscle plays a crucial role in movement, metabolism, and energy homeostasis. As the most metabolically active endocrine organ in the body, it has recently attracted widespread attention. Skeletal muscle possesses the ability to release adipocytokines, bioactive peptides, small molecular metabolites, nucleotides, and other myogenic cell factors; some of which have been shown to be encapsulated within small vesicles, particularly exosomes. These skeletal muscle exosomes (SKM-Exos) are released into the bloodstream and subsequently interact with receptor cell membranes to modulate the physiological and pathological characteristics of various tissues. Therefore, SKM-Exos may facilitate diverse interactions between skeletal muscle and other tissues while also serving as biomarkers that reflect the physiological and pathological states of muscle function. This review delves into the pivotal role and intricate molecular mechanisms of SKM-Exos and its derived miRNAs in the maturation and rejuvenation of skeletal muscle, along with their intercellular signaling dynamics and physiological significance in interfacing with other tissues.

Transient-resting culture after activation enhances the generation of CD8+ stem cell-like memory T cells from peripheral blood mononuclear cells

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (TSCM) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. TSCM cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8+ TSCM cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8+ T cells in a stem memory phenotype (CD95+ CD45RA+ CCR7+) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8+ TSCM cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.

Genome-wide identification of TCP transcription factors and functional role of UrTCP4 in regulating terpenoid indole alkaloids biosynthesis in Uncaria rhynchophylla

Uncaria rhynchophylla is a widely used Chinese herbal medicine known for its terpenoid indole alkaloids (TIAs), which help in treating hypertension. Teosinte branched 1/cycloidea/proliferating cell factors 1/2 (TCP) TFs have been shown to have a role in the growth and development of plants, but the regulation mechanism of UrTCP in the TIAs biosynthesis pathway is yet unknown. In this study, twenty-six UrTCP genes were identified from the genome of U. rhynchophylla, and these genes were classified into three subgroups based on the phylogenetic analysis. UrTCPs from the same group or subgroup share comparable gene structures and conserved motifs. These 26 UrTCP genes were unevenly distributed throughout 22 chromosomes, and gene segmental duplication occurred. The interspecific co-linearity analysis suggested that UrTCP4 may have an important function in evolutionary process. The expression patterns analysis of UrTCP genes by RT-qPCR showed most UrTCP genes had the highest expression in leaves and stem hooks. The results of co-expression analysis and phylogenetic relationships screened UrTCP4 as the most likely participant in TIAs and its precursor synthesis. Furthermore, the subcellular localization of UrTCP4 indicated that it is located in nucleus. Dual luciferase assays revealed the UrTCP4 protein could activate or repress the transcription of UrLAMT and Ur7-DLH in the TIAs synthesis pathway. Finally, Yeast-one-hybrid assays demonstrated that the UrTCP4 protein can bind to the promoters of UrLAMT and Ur7-DLH. Thus, UrTCP4 may be involved in the regulation of the TIAs biosynthesis pathway. This research establishes a groundwork for elucidating the functions of UrTCP gene family in U. rhynchophylla, offering new insights into the regulation of TIAs.

Clinical Study on Mesenchymal Stem Cell Factors Therapy for Alzheimer’s Disease

Background: Alzheimer’s disease (AD), characterized by cognitive decline, lacked effective cures. Mesenchymal stem cell (MSC) factors (MSCFs) offered a new approach by promoting brain tissue repair and modulating immune responses, presenting a promising alternative to AD treatment with minimal risks. Aim: This study aimed to investigate the effects of MSCF on AD and to compare the effects with traditional MSC treatments. Methods: Sixty patients were divided into control and observation groups, with 30 cases in each group. The control group were injected intravenously with 10 mL of MSCs (5.0 × 109 L-1) plus 100 mL normal saline (once every 5 days for six consecutive treatments). The observation group received intramuscular injections of 0.5 mL (1 mL for the first dose) of MSCF (every other day for 15 consecutive treatments). Amyloid-β 42 (Aβ42) and Tau protein concentrations in cerebrospinal fluid were determined by ELISA pretreatment and at 1, 3, and 6 months’ post-treatment. The Clinical Dementia Rating of AD patients was recorded at these intervals to evaluate treatment efficacy. Results: Aβ42 levels increased, and Tau protein levels decreased in both groups. The CDR score dropped post treatment. The total effective rate and clinical cure rate were 86.67% and 6.70% in the control group and 100% and 40% in the observation group, respectively. MSCF and MSCs uniquely impact AD. Conclusion: MSCs contributed to damaged nerve cell repair, new nerve cell differentiation, and the participation of some dormant nerve cells in physiological activity. MSCF offered a small-dose, rapid, and safe treatment with simple operation.

Investigation of the Impact of Magnetic Resonance Imaging-assisted Surgery on Immune Cell Cytokine Levels and Efficacy in Patients with Gliomas

Objectivethis work was to investigate the impact of magnetic resonance imaging (MRI)-assisted surgery on immune cell factors and surgical efficacy in glioma patients, aiming to deepen understanding of surgical effects on immune status and provide evidence for clinical strategies. Materials and Methodsa retrospective analysis of 120 patients treated at Qianjiang Hospital from January 2022 to December 2023 was conducted. Patients underwent traditional craniotomy (control group, CG) or MRI-assisted stereotactic minimally invasive surgery (research group, RG). Immune cell factors, treatment outcomes, cognitive function, daily activities, and adverse reactions (ARs) were compared. Resultspostoperatively, arginine vasopressin (AVP) and β-endorphin (β-EP) levels decreased in both groups, more so in CG (P<0.05). Helper T cell 1 (Th1) levels increased in both, more in controls, while Th2 and Th17 decreased, more in CG (P<0.05). Glasgow coma scale (GCS) scores increased more in the RG, National Institutes of Health Stroke Scale (NIHSS) scores decreased more (P<0.05), and activities of daily living (ADL) scores increased more in the RG (P<0.05). The RG had more effective treatments and fewer ineffective outcomes (P<0.05), and fewer recurrences, metastases, and ARs (P<0.05). ConclusionMRI-assisted surgery enhances immune factors, functional abilities, and shows efficacy with fewer ARs, indicating its clinical value.

BpTCP19 targets BpWRKY53 to negatively regulate jasmonic acid- and dark-induced leaf senescence in Betula platyphylla

TCP (TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1) is a plant-specific transcription factor that has garnered significant attention due to its wide-ranging involvement in the regulation of plant growth or developmental processes. However, the molecular mechanisms through which TCP genes orchestrate leaf senescence have not been extensively elucidated. BpTCP19, a member of the PCF subfamily in Betula platyphylla, and has high homology to AtTCP19. BpTCP19 displayed pronounced downregulation in response to methyl jasmonate (MeJA) and dark treatment. Overexpressing BpTCP19 in Betula platyphylla led to a delay in leaf senescence, resulting in prolonged leaf greenness under both MeJA and dark conditions. Transcriptome analysis revealed that overexpression of BpTCP19 induced alterations in the expression levels of genes linked to cell proliferation, hormone signaling transduction, and leaf senescence, including the early responsive factor BpWRKY53. Furthermore, through Yeast one-hybrid assays and GUS analysis, BpTCP19 was shown to bind to the promoter region of BpWRKY53, suppressing its expression and thereby retarding leaf senescence. This study elucidates the physiological and molecular functions of BpTCP19 as a central transcriptional regulatory module in leaf senescence and provides a potential target gene for delaying leaf senescence by mitigating sensitivity to external aging signals such as Jasmonic acid (JA) and darkness.

Receptors and Host Factors for Enterovirus Infection: Implications for Cancer Therapy.

Enteroviruses, with their diverse clinical manifestations ranging from mild or asymptomatic infections to severe diseases such as poliomyelitis and viral myocarditis, present a public health threat. However, they can also be used as oncolytic agents. This review shows the intricate relationship between enteroviruses and host cell factors. Enteroviruses utilize specific receptors and coreceptors for cell entry that are critical for infection and subsequent viral replication. These receptors, many of which are glycoproteins, facilitate virus binding, capsid destabilization, and internalization into cells, and their expression defines virus tropism towards various types of cells. Since enteroviruses can exploit different receptors, they have high oncolytic potential for personalized cancer therapy, as exemplified by the antitumor activity of certain enterovirus strains including the bioselected non-pathogenic Echovirus type 7/Rigvir, approved for melanoma treatment. Dissecting the roles of individual receptors in the entry of enteroviruses can provide valuable insights into their potential in cancer therapy. This review discusses the application of gene-targeting techniques such as CRISPR/Cas9 technology to investigate the impact of the loss of a particular receptor on the attachment of the virus and its subsequent internalization. It also summarizes the data on their expression in various types of cancer. By understanding how enteroviruses interact with specific cellular receptors, researchers can develop more effective regimens of treatment, offering hope for more targeted and efficient therapeutic strategies.

Exploring HIV-1 Maturation: A New Frontier in Antiviral Development.

HIV-1 virion maturation is an essential step in the viral replication cycle to produce infectious virus particles. Gag and Gag-Pol polyproteins are assembled at the plasma membrane of the virus-producer cells and bud from it to the extracellular compartment. The newly released progeny virions are initially immature and noninfectious. However, once the Gag polyprotein is cleaved by the viral protease in progeny virions, the mature capsid proteins assemble to form the fullerene core. This core, harboring two copies of viral genomic RNA, transforms the virion morphology into infectious virus particles. This morphological transformation is referred to as maturation. Virion maturation influences the distribution of the Env glycoprotein on the virion surface and induces conformational changes necessary for the subsequent interaction with the CD4 receptor. Several host factors, including proteins like cyclophilin A, metabolites such as IP6, and lipid rafts containing sphingomyelins, have been demonstrated to have an influence on virion maturation. This review article delves into the processes of virus maturation and Env glycoprotein recruitment, with an emphasis on the role of host cell factors and environmental conditions. Additionally, we discuss microscopic technologies for assessing virion maturation and the development of current antivirals specifically targeting this critical step in viral replication, offering long-acting therapeutic options.

Studying Retroviral Life Cycles Using Visible Viruses and Live Cell Imaging.

Viruses exploit key host cell factors to accomplish each individual stage of the viral replication cycle. To understand viral pathogenesis and speed the development of new antiviral strategies, high-resolution visualization of virus-host interactions is needed to define where and when these events occur within cells. Here, we review state-of-the-art live cell imaging techniques for tracking individual stages of viral life cycles, focusing predominantly on retroviruses and especially human immunodeficiency virus type1, which is most extensively studied. We describe how visible viruses can be engineered for live cell imaging and how nonmodified viruses can, in some instances, be tracked and studied indirectly using cell biosensor systems. We summarize the ways in which live cell imaging has been used to dissect the retroviral life cycle. Finally, we discuss select challenges for the future including the need for better labeling strategies, increased resolution, and multivariate systems that will allow for the study of full viral replication cycles.

A targeted CRISPR screen identifies ETS1 as a regulator of HIV latency.

HIV latency is a major obstacle for the eradication of HIV. However, molecular mechanisms that restrict proviral expression during therapy are not well understood. Identification of host cell factors that silence HIV would create opportunities for targeting these factors to reverse latency and eliminate infected cells. Our study aimed to explore mechanisms of latency in infected cells by employing a lentiviral CRISPR screen and CRISPR/Cas9 knockout in primary CD4 T cells. These experiments revealed that ETS1 is essential for maintaining HIV latency in primary CD4 T cells and we further confirmed ETS1's role in maintaining HIV latency through CRISPR/Cas9 knockout in CD4 T cells from antiretroviral therapy (ART)-suppressed individuals with HIV. Transcriptomic profiling of ETS1-depleted cells from these individuals identified several host cell pathways involved in viral transcription regulated by ETS1, including the long non-coding RNA MALAT1. Overall, our study demonstrates that ETS1 is a critical regulator of HIV latency, affecting the expression of several cellular genes that directly or indirectly influence HIV expression.

Outcome of Upfront Surgically Resected Patients of Oral Tongue Squamous Cell Carcinoma and Factors Affecting it: Experience from Tertiary Care Facility in North India.

Oral tongue squamous cell carcinoma (OTSCC) is the most aggressive subsite among oral cancers. The poor survival rate has been primarily attributed to high loco-regional recurrence. Two recent developments viz. incorporation depth of invasion (DOI) in American Joint Committee on Cancer (AJCC) TNM 8th edition and elective neck dissection in clinically negative neck have potential to improve survival. We in our study have tried to look at overall survival and factors affecting patients of only OTSCC. 144 patients of OTSCC operated upfront between July 2017 and December 2023 were included in our study. Selective neck dissection was done in all patients with clinically negative neck. T staging was done using both AJCC TNM 7th and 8th edition. Primary objective of the study was to determine the overall survival and factors affecting it. The secondary objectives were to determine the disease-free survival and to look at the effect of forementioned new developments in patients in OTSCC. Mean overall survival and disease-free survival in our study cohort was 48.8 months and 48.3 months respectively in follow up period ranging from 2 months to 75 months. DOI > 10 millimetres and involved margins were factors significantly associated with survival on multivariate analysis. Lymph node metastasis was detected in 32(35.2%) patients out of 91 patients with clinically negative neck and 31(21.6%) of patients were upstaged from T1/T2 in AJCC TNM 7th to T3/T4 according to AJCC TNM 8th edition. The 5-years overall survival of our patients was about 54% with nearly half of our patients presenting in stage III and stage IV. There is need to create awareness in general population as the impact of the new changes will only be seen if patients present at an early stage.

O-135 The consequences of paternal pre-conceptional health for offspring health and pregnancy outcomes: involvement of the germ-cell epigenome and non-germ cell factors

Abstract Study question Paternal health at conception is important for offspring health. The susceptibility windows within the male reproductive tract and the molecular mechanisms are not completely understood. Summary answer Using mouse genetics, (epi)genomics and in-depth phenotyping, we show that the germ-cell epigenome and components of the seminal fluid are critical for paternal intergenerational effects. What is known already Paternal intergenerational effects are highly conserved across mammals and lower organisms including flies and nematodes. In mammals, the sperm epigenome, as well as the composition of the seminal fluid are sensitive to environmental challenges and influence offspring development and adult phenotypic trajectories. The best known of these mechanisms are DNA methylation and small non-coding RNAs in spermatozoa and the balance between inflammatory and tolerogenic cytokines in the seminal fluid. Small non-coding RNAs are mostly acquired by maturing spermatozoa during the epididymal transit from extracellular vesicle of epididymal epithelial origin, and have the potential to influence embryonic development and offspring health. Study design, size, duration We use two paternal paradigms of pre-conceptional challenge in wild-type C57BL6 mice: high-fat diet feeding for 2 weeks and circadian disruption for 4 weeks before conception. The duration of the environmental challenges has been chosen to meet timing of sperm maturation in the mouse (high-fat diet) or to be the minimum sufficient to induce circadian disruption without overt side effects. Exposed males and their offspring have undergone deep metabolic and molecular phenotyping. Participants/materials, setting, methods We used both bulk and single cell (epi)genomics on exposed males as well as male and female offspring. The molecular phenotyping has been instructed by the results of the metabolic phenotyping performed in collaboration with the German Mouse Clinic, the biggest mouse clinic in Europe, using highly standardized and robust procedures. Importantly, we have used a family-based approach for the data analysis, according to which the experimental n is the exposed father. Main results and the role of chance Paternal circadian disruption achieved by four weeks of day-restricted feeding (food only during the light phase), leads to hyperphagia, hyperglycemia, hypercorticosteronemia and partial circadian disruption in unexposed male offspring. The phenotypes are gender-specific and the females feature partial protection from the paternal effects. Mechanistically, mouse seminal fluid contains corticosterone, whose appearance dynamic follows the organismal circadian rhythm and is blunted upon circadian disruption. Offspring phenotypes are sensitive to the levels of corticosterone in the seminal fluid and consequential to impaired placentation, reduced placental efficiency and fetal growth restriction. Paternal exposure to an acute high-fat diet challenge, instead, leads to a 30% penetrant glucose intolerance phenotype exclusively in male offspring. Mechanistically, this is linked to diet-induced mitochondrial dysfunction in somatic and germ cells and a compensatory burst of mitochondrial DNA transcription in spermatozoa. Mitochondrial DNA encoded small non-coding RNAs accumulate in spermatozoa, are inherited at fertilization, and reprogram early embryo transcription towards premature activation of oxidative metabolism, a predisposing factor to adult-onset glucose intolerance. Importantly, phenotypes and mechanisms are conserved across highly characterized human cohorts. These studies strengthen the importance of paternal health at conception for pregnancy and offspring health and identify germ-cell and non-germ cell factors as potential mechanistic mediators. Limitations, reasons for caution Although we have shown for the first time that stress hormones are present in the seminal fluid and relay paternal stress to the offspring and provided the first example of epigenetic inheritance of sperm small non-coding RNAs, mechanisms are still to be completely uncovered. Complementary mechanisms cannot be excluded. Wider implications of the findings Our findings strengthen the importance of paternal health at conception for pregnancy and offspring health. They also provide potential male tract biomarkers and novel strategies to monitor paternal health at conception and preconception lifestyle interventions aimed at improving pregnancy outcomes and preventing the spread of metabolic disorders through epigenetic inheritance. Trial registration number not applicable

Potential of Guava (Psidium guajava L.) as an Additional Therapy for Dengue Fever

Dengue fever has been a global health problem for over five decades, especially in tropical and subtropical countries. It is caused by a viral infection that spreads from mosquito to human. Natural sources have the potency to be an alternative to dengue fever therapy, such as guava (Psidium guajava L.). Its leaves contain several compounds and have several bioactivities. This research aims to explore the potency of guava as an additional therapy for dengue fever patients. The method used a narrative literature review that searches literature in primary data such as national or international journals with the database PubMed or Scopus. The results reported that quercetin is one of the ingredients found in guava that affects dengue fever. It suppresses intracellular replication of dengue virus type 2 (DENV-2) and inhibits ATPase in DENV-4. In addition, quercetin also stimulates stem cell factors in bone marrow stromal cells to produce thrombopoietin which functions to regulate platelet production in the spinal cord. So, guava has the potential and effectiveness to be used as an alternative therapy for dengue fever.

Identification of diagnose related therapeutic targets of Danggui buxue decoction in Parkinson’s disease

BackgroundParkinson’s disease (PD) is the fastest growing neurological disease. Currently, there is no disease-modifying therapy to slow the progression of the disease. Danggui buxue decoction (DBD) is widely used in the clinic because of its therapeutic effect. However, little is known about the molecular mechanism of DBD against PD. This study intends to explore the possible molecular mechanisms involved in DBD treatment of PD based on network pharmacology, and provide potential research directions for future research. MethodsFirstly, the active components and target genes of DBD were screened from the traditional Chinese medicine systems pharmacology (TCMSP), DrugBank and UniProt database. Secondly, target genes of PD were identified from the (GEO) dataset, followed by identification of common target genes of DBD and PD. Thirdly, analysis of protein–protein interaction (PPI), functional enrichment and diagnosis was performed on common target genes, followed by correlation analysis between core target genes, immune cell, miRNAs, and transcription factors (TFs). Finally, molecular docking between core target genes and active components, and real-time PCR were performed. ResultsA total of 72 common target genes were identified between target genes of DBD and target genes of PD. Among which, 11 target genes with potential diagnostic value were further identified, including TP53, AKT1, IL1B, MMP9, NOS3, RELA, MAPK14, HMOX1, TGFB1, NOS2, and ERBB2. The combinations with the best docking binding were identified, including kaempferol-AKT1/HMOX1/NOS2/NOS3, quercetin-AKT1/ERBB2/IL1B/HMOX1/MMP9/TP53/NOS3/TGFB1. Moreover, IL1B and NOS2 respectively positively and negatively correlated with neutrophil and Type 1 T helper cell. Some miRNA-core target gene regulatory pairs were identified, such as hsa-miR-185-5p-TP53/TGFB1/RELA/MAPK14/IL1B/ERBB2/AKT1 and hsa-miR-214-3p-NOS3. These core target genes were significantly enriched in focal adhesion, TNF, HIF-1, and ErbB signaling pathway. ConclusionDiagnostic TP53, AKT1, IL1B, MMP9, NOS3, RELA, MAPK14, HMOX1, TGFB1, NOS2, and ERBB2 may be considered as potential therapeutic targets of DBD in the treatment of PD.

Proteomic insights into the regulatory function of ARID1A in colon cancer cells.

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Coxiella burnetii-containing vacuoles interact with host recycling endosomal proteins Rab11a and Rab35 for vacuolar expansion and bacterial growth.

Coxiella burnetii is a gram-negative obligate intracellular bacterium and a zoonotic pathogen that causes human Q fever. The lack of effective antibiotics and a licensed vaccine for Coxiella in the U.S. warrants further research into Coxiella pathogenesis. Within the host cells, Coxiella replicates in an acidic phagolysosome-like vacuole termed Coxiella-containing vacuole (CCV). Previously, we have shown that the CCV pH is critical for Coxiella survival and that the Coxiella Type 4B secretion system regulates CCV pH by inhibiting the host endosomal maturation pathway. However, the trafficking pattern of the 'immature' endosomes in Coxiella- infected cells remained unclear. We transfected HeLa cells with GFP-tagged Rab proteins and subsequently infected them with mCherry-Coxiella to visualize Rab protein localization. Infected cells were immunostained with anti-Rab antibodies to confirm the Rab localization to the CCV, to quantitate Rab11a and Rab35- positive CCVs, and to quantitate total recycling endosome content of infected cells. A dual-hit siRNA mediated knockdown combined with either immunofluorescent assay or an agarose-based colony-forming unit assay were used to measure the effects of Rab11a and Rab35 knockdown on CCV area and Coxiella intracellular growth. The CCV localization screen with host Rab proteins revealed that recycling endosome-associated proteins Rab11a and Rab35 localize to the CCV during infection, suggesting that CCV interacts with host recycling endosomes during maturation. Interestingly, only a subset of CCVs were Rab11a or Rab35-positive at any given time point. Quantitation of Rab11a/Rab35-positive CCVs revealed that while Rab11a interacts with the CCV more at 3 dpi, Rab35 is significantly more prevalent at CCVs at 6 dpi, suggesting that the CCV preferentially interacts with Rab11a and Rab35 depending on the stage of infection. Furthermore, we observed a significant increase in Rab11a and Rab35 fluorescent intensity in Coxiella-infected cells compared to mock, suggesting that Coxiella increases the recycling endosome content in infected cells. Finally, siRNA-mediated knockdown of Rab11a and Rab35 resulted in significantly smaller CCVs and reduced Coxiella intracellular growth, suggesting that recycling endosomal Rab proteins are essential for CCV expansion and bacterial multiplication. Our data, for the first time, show that the CCV dynamically interacts with host recycling endosomes for Coxiella intracellular survival and potentially uncovers novel host cell factors essential for Coxiella pathogenesis.

Identifications of novel host cell factors that interact with the receptor-binding domain of the SARS-CoV-2 spike protein

SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent, but integrin- and clathrin-dependent manner, and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9 and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to entry of SARS-CoV-2.