Search

Abstract Background Primary ovarian insufficiency (POI) is an early decline in ovarian function that leads to ovarian failure. Conventional treatments for POI are inadequate, and treatments based on mesenchymal stem cells (MSCs) have emerged as an option. However, the lack of consideration of the estrogen niche in ovarian tissue significantly reduces the therapeutic efficacy, with an unclear mechanism in the MSCs in POI treatment. Furthermore, the disruption of circadian rhythm associated with POI has not been previously addressed. Methods Conditioned medium (CM) and estradiol-conditioned medium (E2-CM) were generated from estrogen receptor positive MSCs (ER+pcMSCs). Chemotherapy-induced POI models were established using C57BL/6 mice (in vivo) and KGN cells (in vitro) treated with cyclophosphamide (CTX) or 4-hydroperoxycyclophosphamide (4-OOH-CP). Gene/protein expressions were detected using RT-qPCR, Western blotting, and immunohistochemistry assays. Locomotor activity was monitored for behavioral circadian rhythmicity. Cytokine arrays and miRNA analysis were conducted to analyze potential factors within CM/E2-CM. Results The secretome of ER+pcMSCs (CM and E2-CM) significantly reduced the CTX-induced defects in ovarian folliculogenesis and circadian rhythm. CM/E2-CM also reduced granulosa cell apoptosis and rescued angiogenesis in POI ovarian tissues. E2-CM had a more favorable effect than the CM. Notably, ER+pcMSC secretome restored CTX-induced circadian rhythm defects, including the gene expressions associated with the ovarian circadian clock (e.g., Rora, E4bp4, Rev-erbα, Per2 and Dbp) and locomotor activity. Additionally, the cytokine array analysis revealed a significant increase in cytokines and growth factors associated with immunomodulation and angiogenesis, including angiogenin. Neutralizing the angiogenin in CM/E2-CM significantly reduced its ability to promote HUVEC tube formation in vitro. Exosomal miRNA analysis revealed the miRNAs involved in targeting the genes associated with POI rescue (PTEN and PDCD4), apoptosis (caspase-3, BIM), estrogen synthesis (CYP19A1), ovarian clock regulation (E4BP4, REV-ERBα) and fibrosis (COL1A1). Conclusion This study is the first to demonstrate that, in considering the estrogen niche in ovarian tissue, an estrogen-priming ER+pcMSC secretome achieved ovarian regeneration and restored the circadian rhythm in a CTX-induced POI mouse model. The potential factors involved include angiogenin and exosomal miRNAs in the ER+pcMSC secretome. These findings offer insights into potential stem cell therapies for chemotherapy-induced POI and circadian rhythm disruption.

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC–derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system–generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.

Abstract Background Cell composition deconvolution (CCD) is a type of bioinformatic task to estimate the cell fractions from bulk gene expression profiles, such as RNA-seq. Many CCD models were developed to perform linear regression analysis using reference gene expression signatures of distinct cell types. Reference gene expression signatures could be generated from cell-specific gene expression profiles, such as scRNA-seq. However, the batch effects and dropout events frequently observed across scRNA-seq datasets have limited the performances of CCD methods. Methods We developed a deep neural network (DNN) model, HASCAD, to predict the cell fractions of up to 15 immune cell types. HASCAD was trained using the bulk RNA-seq simulated from three scRNA-seq datasets that have been normalized by using a Harmony-Symphony based strategy. Mean square error and Pearson correlation coefficient were used to compare the performance of HASCAD with those of other widely used CCD methods. Two types of datasets, including a set of simulated bulk RNA-seq, and three human PBMC RNA-seq datasets, were arranged to conduct the benchmarks. Results HASCAD is useful for the investigation of the impacts of immune cell heterogeneity on the therapeutic effects of immune checkpoint inhibitors, since the target cell types include the ones known to play a role in anti-tumor immunity, such as three subtypes of CD8 T cells and three subtypes of CD4 T cells. We found that the removal of batch effects in the reference scRNA-seq datasets could benefit the task of CCD. Our benchmarks showed that HASCAD is more suitable for analyzing bulk RNA-seq data, compared with the two widely used CCD methods, CIBERSORTx and quanTIseq. We applied HASCAD to analyze the liver cancer samples of TCGA-LIHC, and found that there were significant associations of the predicted abundance of Treg and effector CD8 T cell with patients’ overall survival. Conclusion HASCAD could predict the cell composition of the PBMC bulk RNA-seq and classify the cell type from pure bulk RNA-seq. The model of HASCAD is available at https://github.com/holiday01/HASCAD .

The passion fruit, Passiflora edulis, recognized for its rich nutritional properties, has long been used for its varied ethnobotanical applications. This study investigates the therapeutic potential of P. edulis var. Tainung No. 1 rind extracts by examining their polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes such as elastase, tyrosinase, and hyaluronidase, and their ability to inhibit bacterial growth, single-stranded DNA-binding protein (SSB), and their cytotoxic effects on oral carcinoma cells. The acetone extract from the rind exhibited the highest levels of TPC, TFC, anti-SSB, and antibacterial activities. The antibacterial effectiveness of the acetone-extracted rind was ranked as follows: Escherichia coli > Pseudomonas aeruginosa > Staphylococcus aureus. A titration curve for SSB inhibition showed an IC50 value of 313.2 μg/mL, indicating the potency of the acetone extract in inhibiting SSB. It also significantly reduced the activity of enzymes associated with skin aging, particularly tyrosinase, with a 54.5% inhibition at a concentration of 100 μg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis tentatively identified several major bioactive compounds in the acetone extract, including stigmast-5-en-3-ol, vitamin E, palmitic acid, stigmasterol, linoleic acid, campesterol, and octadecanoic acid. Molecular docking studies suggested some of these compounds as potential inhibitors of tyrosinase and SSB. Furthermore, the extract demonstrated anticancer potential against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation and inducing apoptosis. These results underscore the potential of P. edulis (Tainung No. 1) rind as a promising candidate for anti-skin aging, antibacterial, and anticancer applications, meriting further therapeutic investigation.

The tamarillo, or Solanum betaceum, recognized for its comprehensive nutritional profile, has long been valued for its diverse ethnobotanical uses. This study delves into the potential therapeutic applications of S. betaceum by analyzing its polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes like elastase, tyrosinase, and hyaluronidase, and its cytotoxic effects on oral carcinoma cells. Extracts from the seeds, pulp, and peel of red and yellow fruits were prepared using methanol, ethanol, and acetone. The highest TPC was found in the methanol extract from red fruit seeds (9.89 mg GAE/g), and the highest TFC was found in the methanol extract of yellow fruit peel (3.02 mg QUE/g). Some of these extracts significantly inhibited skin aging-associated enzymes with the red fruit seed extract (100 μg/mL) showing up to 50.4% inhibition of tyrosinase. Additionally, the red fruit seed extract obtained using methanol demonstrated potential anticancer effects against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation as well as inducing apoptosis. These results underscore the potential of S. betaceum fruit extracts, especially from red fruit seeds, as promising agents for anti-skin aging and anticancer applications, meriting further exploration for therapeutic uses.

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9’s ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.

Abstract Background Many studies have utilized computational methods, including cell composition deconvolution (CCD), to correlate immune cell polarizations with the survival of cancer patients, including those with hepatocellular carcinoma (HCC). However, currently available cell deconvolution estimated (CDE) tools do not cover the wide range of immune cell changes that are known to influence tumor progression. Results A new CCD tool, HCCImm, was designed to estimate the abundance of tumor cells and 16 immune cell types in the bulk gene expression profiles of HCC samples. HCCImm was validated using real datasets derived from human peripheral blood mononuclear cells (PBMCs) and HCC tissue samples, demonstrating that HCCImm outperforms other CCD tools. We used HCCImm to analyze the bulk RNA‐seq datasets of The Cancer Genome Atlas (TCGA)‐liver hepatocellular carcinoma (LIHC) samples. We found that the proportions of memory CD8+ T cells and Tregs were negatively associated with patient overall survival (OS). Furthermore, the proportion of naïve CD8+ T cells was positively associated with patient OS. In addition, the TCGA‐LIHC samples with a high tumor mutational burden had a significantly high abundance of nonmacrophage leukocytes. Conclusions HCCImm was equipped with a new set of reference gene expression profiles that allowed for a more robust analysis of HCC patient expression data. The source code is provided at https://github.com/holiday01/HCCImm.

Abstract Background The distribution of resources can affect animal range sizes, which in turn may alter infectious disease dynamics in heterogenous environments. The risk of pathogen exposure or the spatial extent of outbreaks may vary with host range size. This study examined the range sizes of herbivorous anthrax host species in two ecosystems and relationships between spatial movement behavior and patterns of disease outbreaks for a multi-host environmentally transmitted pathogen. Methods We examined range sizes for seven host species and the spatial extent of anthrax outbreaks in Etosha National Park, Namibia and Kruger National Park, South Africa, where the main host species and outbreak sizes differ. We evaluated host range sizes using the local convex hull method at different temporal scales, within-individual temporal range overlap, and relationships between ranging behavior and species contributions to anthrax cases in each park. We estimated the spatial extent of annual anthrax mortalities and evaluated whether the extent was correlated with case numbers of a given host species. Results Range size differences among species were not linearly related to anthrax case numbers. In Kruger the main host species had small range sizes and high range overlap, which may heighten exposure when outbreaks occur within their ranges. However, different patterns were observed in Etosha, where the main host species had large range sizes and relatively little overlap. The spatial extent of anthrax mortalities was similar between parks but less variable in Etosha than Kruger. In Kruger outbreaks varied from small local clusters to large areas and the spatial extent correlated with case numbers and species affected. Secondary host species contributed relatively few cases to outbreaks; however, for these species with large range sizes, case numbers positively correlated with outbreak extent. Conclusions Our results provide new information on the spatiotemporal structuring of ranging movements of anthrax host species in two ecosystems. The results linking anthrax dynamics to host space use are correlative, yet suggest that, though partial and proximate, host range size and overlap may be contributing factors in outbreak characteristics for environmentally transmitted pathogens.

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32’s DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA metabolism by binding and stabilizing single-stranded DNA (ssDNA) intermediates. Through their multifaceted roles in DNA replication, recombination, repair, replication restart, and other cellular processes, SSB emerges as a central player in maintaining genomic integrity. These attributes collectively position SSBs as essential guardians of genomic integrity, establishing interactions with an array of distinct proteins. Unlike Escherichia coli, which contains only one type of SSB, some bacteria have two paralogous SSBs, referred to as SsbA and SsbB. In this study, we identified Staphylococcus aureus SsbA (SaSsbA) as a fresh addition to the roster of the anticancer drug 5-fluorouracil (5-FU) binding proteins, thereby expanding the ambit of the 5-FU interactome to encompass this DNA replication protein. To investigate the binding mode, we solved the complexed crystal structure with 5-FU at 2.3 Å (PDB ID 7YM1). The structure of glycerol-bound SaSsbA was also determined at 1.8 Å (PDB ID 8GW5). The interaction between 5-FU and SaSsbA was found to involve R18, P21, V52, F54, Q78, R80, E94, and V96. Based on the collective results from mutational and structural analyses, it became evident that SaSsbA’s mode of binding with 5-FU diverges from that of SaSsbB. This complexed structure also holds the potential to furnish valuable comprehension regarding how 5-FU might bind to and impede analogous proteins in humans, particularly within cancer-related signaling pathways. Leveraging the information furnished by the glycerol and 5-FU binding sites, the complexed structures of SaSsbA bring to the forefront the potential viability of several interactive residues as potential targets for therapeutic interventions aimed at curtailing SaSsbA activity. Acknowledging the capacity of microbiota to influence the host’s response to 5-FU, there emerges a pressing need for further research to revisit the roles that bacterial and human SSBs play in the realm of anticancer therapy.

Generic approach for rapid prototyping is essential for the progress of synthetic biology. Here the authors modify the cell-free translation system to control protein aggregation and folding and validate the approach by using single conditions for prototyping of various disulfide-constrained polypeptides.

Measurement(s) RNA-Seq • RNA Technology Type(s) Illumina HiSeq. 2500 • RNA sequencing Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.15022521

Abstract Background Small blood stem cells (SB cells), isolated from human peripheral blood, demonstrated the ability to benefit bone regeneration and osseointegration. The primary goal of our study is to examine the safety and tolerability of SB cells in dental implantation for human patients with severe bone defects. Methods Nine patients were enrolled and divided into three groups with SB cell treatment doses of 1 × 105, 1 × 106, and 1 × 107 SB cells, and then evaluated by computed tomography (CT) scans to assess bone mineral density (BMD) by Hounsfield units (HU) scoring. Testing was conducted before treatment and on weeks 4, 6, 8, and 12 post dental implantation. Blood and comprehensive chemistry panel testing were also performed. Results No severe adverse effects were observed for up to 6-month trial. Grade 1 leukocytosis, anemia, and elevated liver function were observed, but related with the patient’s condition or the implant treatment itself and not the transplantation of SB cells. The levels of cytokines and chemokines were detected by a multiplex immunological assay. Elevated levels of eotaxin, FGF2, MCP-1, MDC, and IL17a were found among patients who received SB cell treatment. This observation suggested SB cells triggered cytokines and chemokines for local tissue repair. To ensure the efficacy of SB cells in dental implantation, the BMD and maximum stresses via stress analysis model were measured through CT scanning. All patients who suffered from severe bone defect showed improvement from D3 level to D1 or D2 level. The HU score acceleration can be observed by week 2 after guided bone regeneration (GBR) and prior to dental implantation. Conclusions This phase I study shows that treatment of SB cells for dental implantation is well tolerated with no major adverse effects. The use of SB cells for accelerating the osseointegration in high-risk dental implant patients warrants further phase II studies. Trial registration Taiwan Clinical Trial Registry ( SB-GBR001 ) and clinical trial registry of the United States ( NCT04451486 ).

Abstract Since 2013, a high incidence of bilateral symmetrical alopecia has been observed in free-ranging Formosan macaques (Macaca cyclopis) in Mt. Longevity, Taiwan. We hypothesized that stress induces alopecia in this population. To verify our hypothesis, we evaluated the histopathological characteristics of skin biopsy and used a validated enzyme immunoassay (EIA) for fecal glucocorticoid metabolite (FGM) analysis, which act as an indicator of stress experienced by the individual. Follicular densities were lower (2.1–3.0 mm2) in individuals with symmetrical alopecia than in those with normal hair conditions (4.7 mm2). Furthermore, anagen to catagen/telogen ratios were lower in individuals with alopecia (0–1.4) than in those with normal hair (4.0). The histopathological characteristics of alopecia were similar to those of telogen effluvium, which indicates that stress is one of the possible etiologies. On the basis of the analytical and biological validation of EIAs for FGM analysis, 11β-hydroxyetiocholanolone was considered suitable for monitoring adrenocortical activity in both sexes of Formosan macaques. The mean concentrations (standard error; sample size) of 11β-hydroxyetiocholanolone were 2.02 (0.17; n = 10) and 1.41 (0.10; n = 31) μg/g for individuals with and without alopecia, respectively. Furthermore, the results of logistic regression analysis show that 11β-hydroxyetiocholanolone (p = 0.012) concentration was positively associated with alopecia. Thus, stress was the most likely to trigger symmetrical alopecia in Formosan macaques in Mt. Longevity. Although stress can decrease the fitness of an individual, considering the population status of Formosan macaques in Taiwan is stable and alopecia was only observed in our study area, which is isolated from other populations, the impact on the total population of Formosan macaque in Taiwan is limited. Nonetheless, stress-induced immunosuppression and alopecia might affect the local abundance and increase zoonosis risk due to frequent human–macaque contact in Mt. Longevity. Future studies are suggested to focus on the causative factor of stress and the effects of stress and alopecia on the health and welfare in the Formosan macaques.

Abstract Mesenchymal stem/stromal cells (MSCs) are a promising resource for cell-based therapy because of their high immunomodulation ability, tropism towards inflamed and injured tissues, and their easy access and isolation. Currently, there are more than 1200 registered MSC clinical trials globally. However, a lack of standardized methods to characterize cell safety, efficacy, and biodistribution dramatically hinders the progress of MSC utility in clinical practice. In this review, we summarize the current state of MSC-based cell therapy, focusing on the systemic safety and biodistribution of MSCs. MSC-associated risks of tumor initiation and promotion and the underlying mechanisms of these risks are discussed. In addition, MSC biodistribution methodology and the pharmacokinetics and pharmacodynamics of cell therapies are addressed. Better understanding of the systemic safety and biodistribution of MSCs will facilitate future clinical applications of precision medicine using stem cells.

The carnivorous pitcher plants of the genus Nepenthes exhibit many ethnobotanical uses, including treatments of stomachache and fever. In this study, we prepared different extracts from the pitcher, stem, and leaf extracts of Nepenthes miranda obtained using 100% methanol and analyzed their inhibitory effects on recombinant single-stranded DNA-binding protein (SSB) from Klebsiella pneumoniae (KpSSB). SSB is essential for DNA replication and cell survival and thus an attractive target for potential antipathogen chemotherapy. Different extracts prepared from Sinningia bullata, a tuberous member of the flowering plant family Gesneriaceae, were also used to investigate anti-KpSSB properties. Among these extracts, the stem extract of N. miranda exhibited the highest anti-KpSSB activity with an IC50 value of 15.0 ± 1.8 μg/mL. The cytotoxic effects of the stem extract of N. miranda on the survival and apoptosis of the cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were also demonstrated and compared. Based on collective data, the cytotoxic activities of the stem extract at a concentration of 20 μg/mL followed the order Ca9-22 > CAL27 > PC9 > 4T1 > B16F10 cells. The stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation. In addition, incubation with this extract at a concentration of 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 29.2% in the Ca9-22 cells; in other words, the stem extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest. Through gas chromatography–mass spectrometry, the 16 most abundant compounds in the stem extract of N. miranda were tentatively identified. The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis, and their docking scores were compared. The binding capacity of these compounds was in the order sitosterol > hexadecanoic acid > oleic acid > plumbagin > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione > methyl α-d-galactopyranoside > 3-methoxycatechol > catechol > pyrogallol > hydroxyhydroquinone; thus, sitosterol might exhibit the greatest inhibitory capacity against KpSSB among the selected compounds. Overall, these results may indicate the pharmacological potential of N. miranda for further therapeutic applications.

Background and ObjectivesThe novel coronavirus disease 2019 (COVID-19) has been a pandemic health issue in 30 January 2020. The mortality rate is as high as 50% in critically ill patients. Stem cell therapy is effective for those who are refractory to standard treatments. However, the immune responses that underlie stem cell therapy have not been well reported, particularly, in patients associated with moderate to severe acute respiratory distress syndrome (ARDS).MethodsOn Days 0 and 4, an intravenous infusion of 2 × 107 placenta-derived mesenchymal stem cells (pcMSCs) (MatriPlax) were administered to five severe COVID-19 patients refractory to current standard therapies. Peripheral blood inflammatory markers and immune profiles were determined by multi-parameter flow cytometry and studied at Days 0, 4, and 8. Clinical outcomes were also observed.ResultsNone of the pc-MSC treated patients experienced 28-day mortality compared with the control group and showed a significant improvement in the PaO2/FiO2 ratio, Murray’s lung injury scores, reduction in serum ferritin, lactate dehydrogenase (LDH), and C-reactive protein (CRP) levels. The cytokine profiles also showed a reduction in IL-1β, IFN-γ, IL-2, and IL-6, and an increase in IL-13 and IL-5 type 2 cytokines within 7 days of therapy. Lymphopenia was also significantly improved after 7 days of treatment. Immune cell profiles showed an increase in the proportions of CD4+ T cells (namely, CD4+ naïve T cells and CD4+ memory T cell subtypes), Treg cells, CD19+ B cells (namely, CD19+ naïve B cells, CD27+ switched B cell subtypes) and dendritic cells, and a significant decrease in the proportion of CD14+ monocytes (namely, CD16- classical and CD16+ non-classical subtypes), and plasma/plasmablast cells. No adverse effects were seen at the serial follow-up visits for 2 months after initial therapy.Conclusionpc-MSCs therapy suppressed hyper-inflammatory states of the innate immune response to COVID-19 infection by increasing Treg cells, decreasing monocytes and plasma/plasmablast cells, and promoting CD4+ T cells and CD19+ B cells toward adaptive immune responses in severely critically ill COVID-19 patients with moderate to severe ARDS, especially those who were refractory to current standard care and immunosuppressive therapies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive upper and lower motor neuron (MN) degeneration with unclear pathology. The worldwide prevalence of ALS is approximately 4.42 per 100,000 populations, and death occurs within 3–5 years after diagnosis. However, no effective therapeutic modality for ALS is currently available. In recent years, cellular therapy has shown considerable therapeutic potential because it exerts immunomodulatory effects and protects the MN circuit. However, the safety and efficacy of cellular therapy in ALS are still under debate. In this review, we summarize the current progress in cellular therapy for ALS. The underlying mechanism, current clinical trials, and the pros and cons of cellular therapy using different types of cell are discussed. In addition, clinical studies of mesenchymal stem cells (MSCs) in ALS are highlighted. The summarized findings of this review can facilitate the future clinical application of precision medicine using cellular therapy in ALS.

Abstract The latitudinal diversity gradient (LDG) is an established macroecological pattern, but is poorly studied in microbial organisms, particularly parasites. In this study, we tested whether latitude, elevation, and host species predicted patterns of prevalence, alpha diversity, and community turnover of hemosporidian parasites. We expected parasite diversity to decrease with latitude, alongside the diversity of their hosts and vectors. Similarly, we expected infection prevalence to decrease with latitude as vector abundances decrease. Lastly, we expected parasite community turnover to increase with latitudinal distance and to be higher between rather than within host species. We tested these hypotheses by screening blood and tissue samples of three closely related avian species in a clade of North American songbirds (Turdidae: Catharus, n = 466) across 17.5° of latitude. We used a nested PCR approach to identify parasites in hemosporidian genera that are transmitted by different dipteran vectors. Then, we implemented linear‐mixed effects and generalized dissimilarity models to evaluate the effects of latitude, elevation, and host species on parasite metrics. We found high diversity of hemosporidian parasites in Catharus thrushes (n = 44 lineages) but no evidence of latitudinal gradients in alpha diversity or prevalence. Parasites in the genus Leucocytozoon were most prevalent and lineage rich in this study system; however, there was limited turnover with latitude and host species. Contrastingly, Plasmodium parasites were less prevalent and diverse than Leucocytozoon parasites, yet communities turned over at a higher rate with latitude and host species. Leucocytozoon communities were skewed by the dominance of one or two highly prevalent lineages with broad latitudinal distributions. The few studies that evaluate the hemosporidian LDG do not find consistent patterns of prevalence and diversity, which makes it challenging to predict how they will respond to global climate change.

Exposure and immunity to generalist pathogens differ among host species and vary across spatial scales. Anthrax, caused by a multi-host bacterial pathogen, Bacillus anthracis, is enzootic in Kruger National Park (KNP), South Africa and Etosha National Park (ENP), Namibia. These parks share many of the same potential host species, yet the main anthrax host in one (greater kudu (Tragelaphus strepsiceros) in KNP and plains zebra (Equus quagga) in ENP) is only a minor host in the other. We investigated species and spatial patterns in anthrax mortalities, B. anthracis exposure, and the ability to neutralize the anthrax lethal toxin to determine if observed host mortality differences between locations could be attributed to population-level variation in pathogen exposure and/or immune response. Using serum collected from zebra and kudu in high and low incidence areas of each park (18- 20 samples/species/area), we estimated pathogen exposure from anti-protective antigen (PA) antibody response using enzyme-linked immunosorbent assay (ELISA) and lethal toxin neutralization with a toxin neutralization assay (TNA). Serological evidence of pathogen exposure followed mortality patterns within each system (kudus: 95% positive in KNP versus 40% in ENP; zebras: 83% positive in ENP versus 63% in KNP). Animals in the high-incidence area of KNP had higher anti-PA responses than those in the low-incidence area, but there were no significant differences in exposure by area within ENP. Toxin neutralizing ability was higher for host populations with lower exposure prevalence, i.e., higher in ENP kudus and KNP zebras than their conspecifics in the other park. These results indicate that host species differ in their exposure to and adaptive immunity against B. anthracis in the two parks. These patterns may be due to environmental differences such as vegetation, rainfall patterns, landscape or forage availability between these systems and their interplay with host behavior (foraging or other risky behaviors), resulting in differences in exposure frequency and dose, and hence immune response.

Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

Abstract Background To facilitate the investigation of the pathogenic roles played by various immune cells in complex tissues such as tumors, a few computational methods for deconvoluting bulk gene expression profiles to predict cell composition have been created. However, available methods were usually developed along with a set of reference gene expression profiles consisting of imbalanced replicates across different cell types. Therefore, the objective of this study was to create a new deconvolution method equipped with a new set of reference gene expression profiles that incorporate more microarray replicates of the immune cells that have been frequently implicated in the poor prognosis of cancers, such as T helper cells, regulatory T cells and macrophage M1/M2 cells. Methods Our deconvolution method was developed by choosing ε-support vector regression (ε-SVR) as the core algorithm assigned with a loss function subject to the L1-norm penalty. To construct the reference gene expression signature matrix for regression, a subset of differentially expressed genes were chosen from 148 microarray-based gene expression profiles for 9 types of immune cells by using ANOVA and minimizing condition number. Agreement analyses including mean absolute percentage errors and Bland-Altman plots were carried out to compare the performances of our method and CIBERSORT. Results In silico cell mixtures, simulated bulk tissues, and real human samples with known immune-cell fractions were used as the test datasets for benchmarking. Our method outperformed CIBERSORT in the benchmarks using in silico breast tissue-immune cell mixtures in the proportions of 30:70 and 50:50, and in the benchmark using 164 human PBMC samples. Our results suggest that the performance of our method was at least comparable to that of a state-of-the-art tool, CIBERSORT. Conclusions We developed a new cell composition deconvolution method and the implementation was entirely based on the publicly available R and Python packages. In addition, we compiled a new set of reference gene expression profiles, which might allow for a more robust prediction of the immune cell fractions from the expression profiles of cell mixtures. The source code of our method could be downloaded from https://github.com/holiday01/deconvolution-to-estimate-immune-cell-subsets.

Vitiligo is a chronic autoimmune depigmenting skin disorder characterized by patches of the skin losing functional melanocytes. Multiple combinatorial factors are involved in disease development, among which immune T cells play a prominent role. The immune cells implicated in melanocyte destruction through adaptive immunity include CD8+ cytotoxic T cells and regulatory T cells, and aberrantly activated skin-resident memory T cells also play a role in melanocyte destruction. Over the past several years, major progress in understanding vitiligo pathogenesis has led to the development of targeted therapies. Janus kinase (JAK) inhibitors, which share the similar mechanism that autoactivates CD8+ T cells in chronic inflammatory diseases, have been reported to have therapeutic significance in vitiligo. Recently, immunomodulatory therapeutic interventions in vitiligo have been emerging. Mesenchymal stem cells (MSCs) regulate cytokine secretion and the balance of T-cell subsets, which makes them a promising cell-based treatment option for autoimmune diseases. The induction of MSC-mediated immunomodulation is complicated and occurs by contact-dependent mechanisms and soluble extracellular vesicle (EV) mediators. EVs released from MSCs contain various growth factors and cytokines with anti-inflammatory effects in the skin immune response. Here, we summarize and discuss the progress to date in targeted therapies that immunomodulate the niche environment of vitiligo, from the clinical trial of JAK inhibitors to the potential of MSCs and MSC-EVs. The available information was collected to highlight the need for further research into the treatment of vitiligo.

The Staphylococcus aureus SsbA protein (SaSsbA) is a single-stranded DNA-binding protein (SSB) that is categorically required for DNA replication and cell survival, and it is thus an attractive target for potential antipathogen chemotherapy. In this study, we prepared the stem extract of Sarracenia purpurea obtained from 100% acetone to investigate its inhibitory effect against SaSsbA. In addition, the cytotoxic effects of this extract on the survival, apoptosis, proliferation, and migration of B16F10 melanoma cells were also examined. Initially, myricetin, quercetin, kaempferol, dihydroquercetin, dihydrokaempferol, rutin, catechin, β-amyrin, oridonin, thioflavin T, primuline, and thioflavin S were used as possible inhibitors against SaSsbA. Of these compounds, dihydrokaempferol and oridonin were capable of inhibiting the ssDNA-binding activity of SaSsbA with respective IC50 values of 750 ± 62 and 2607 ± 242 μM. Given the poor inhibition abilities of dihydrokaempferol and oridonin, we screened the extracts of S. purpurea, Nepenthes miranda, and Plinia cauliflora for SaSsbA inhibitors. The stem extract of S. purpurea exhibited high anti-SaSsbA activity, with an IC50 value of 4.0 ± 0.3 μg/mL. The most abundant compounds in the stem extract of S. purpurea were identified using gas chromatography–mass spectrometry. The top five most abundant contents in this extract were driman-8,11-diol, deoxysericealactone, stigmast-5-en-3-ol, apocynin, and α-amyrin. Using the MOE-Dock tool, the binding modes of these compounds, as well as dihydrokaempferol and oridonin, to SaSsbA were elucidated, and their binding energies were also calculated. Based on the S scores, the binding capacity of these compounds was in the following order: deoxysericealactone > dihydrokaempferol > apocynin > driman-8,11-diol > stigmast-5-en-3-ol > oridonin > α-amyrin. Incubation of B16F10 cells with the stem extract of S. purpurea at a concentration of 100 μg/mL caused deaths at the rate of 76%, reduced migration by 95%, suppressed proliferation and colony formation by 99%, and induced apoptosis, which was observed in 96% of the B16F10 cells. Overall, the collective data in this study indicate the pharmacological potential of the stem extract of S. purpurea for further medical applications.

The mechanism on how extracellular matrix (ECM) cooperates with niche growth factors and oxygen tension to regulate the self-renewal of embryonic germline stem cells (GSCs) still remains unclear. Lacking of an appropriate in vitro cell model dramatically hinders the progress. Herein, using a serum-free culture system, we demonstrated that ECM laminin cooperated with hypoxia and insulin-like growth factor 1 receptor (IGF-1R) to additively maintain AP activity and Oct-4 expression of AP+GSCs. We found the laminin receptor CD49f expression in d2 testicular GSCs that were surrounded by laminin. Laminin and hypoxia significantly increased the GSC stemness-related genes, including Hif-2α, Oct-4, IGF-1R, and CD49f. Cotreatment of IGF-1 and laminin additively increased the expression of IGF-IR, CD49f, Hif-2α, and Oct-4. Conversely, silencing IGF-1R and/or CD49f decreased the expression of Hif-2α and Oct-4. The underlying mechanism involved CD49f/IGF1R-(PI3K/AKT)-Hif-2α signaling loop, which in turn maintains Oct-4 expression, symmetric self-renewal, and cell migration. These findings reveal the additive niche laminin/IGF-IR network during early GSC development.

Chronic pain is an undertreated epidemic affecting quality of life in at least 20% of the global population, and CNS-related side effects, tolerance, and addiction are common features of current medications. α-Conotoxin Vc1.1 potently elicits prolonged analgesia in preclinical chronic constriction injury and chronic visceral hypersensitivity models of neuropathic pain. A backbone-cyclized variant, cVc1.1, exhibits superior in vitro stability and is orally active, but its in vivo half-life and disposition, both critical in informing drug candidate progression, remain unexplored. Here, we investigate the pharmacological influence of the peptidic bridge differentiating linear and cyclic Vc1.1 in various preclinical PK/PD rodent models. While previous in vitro studies had indicated cyclization conferred increased stability for cVc1.1, in vivo the peptides exhibited similar half-lives and oral bioavailabilities. The ratios of free drug exposure metrics (Cmax × fu,p and AUC0-inf × fu,p) following oral dosing vs. their respective in vitro IC50s at the GABAB receptor were comparable for Vc1.1 and cVc1.1, indicating similar drug efficiency indexes. MALDI imaging, radiolabel, and LC-MS biodistribution studies of cVc1.1 in rodents demonstrated that the intact cyclopeptide and several metabolites persist in the GI tract for at least 4 h, long after the plasma levels of the intact peptide had fallen below the target IC50. Biodistribution analyses of IV administered 125I-labelled cVc1.1 revealed accumulation primarily in the kidneys consistent with renal elimination, and combined with insignificant uptake in brain, suggested a low likelihood of CNS-related side effects.

Disease outbreaks are a consequence of interactions among the three components of a host–parasite system: the infectious agent, the host and the environment. While virulence and transmission are widely investigated, most studies of parasite life-history trade-offs are conducted with theoretical models or tractable experimental systems where transmission is standardized and the environment controlled. Yet, biotic and abiotic environmental factors can strongly affect disease dynamics, and ultimately, host–parasite coevolution. Here, we review research on how environmental context alters virulence–transmission relationships, focusing on the off-host portion of the parasite life cycle, and how variation in parasite survival affects the evolution of virulence and transmission. We review three inter-related ‘approaches’ that have dominated the study of the evolution of virulence and transmission for different host–parasite systems: (i) evolutionary trade-off theory, (ii) parasite local adaptation and (iii) parasite phylodynamics. These approaches consider the role of the environment in virulence and transmission evolution from different angles, which entail different advantages and potential biases. We suggest improvements to how to investigate virulence–transmission relationships, through conceptual and methodological developments and taking environmental context into consideration. By combining developments in life-history evolution, phylogenetics, adaptive dynamics and comparative genomics, we can improve our understanding of virulence–transmission relationships across a diversity of host–parasite systems that have eluded experimental study of parasite life history.

Abstract Background The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear. Methods HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo. Results We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells. Conclusions The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b–mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

The carnivorous pitcher plant Sarracenia purpurea exhibits many ethnobotanical uses, including the treatments of type 2 diabetes and tuberculosis-like symptoms. In this study, we prepared different extracts from the leaves (pitchers), stems, and roots of S. purpurea and investigated their antioxidant and anticancer properties. To evaluate the extraction efficiency, we individually used different solvents, namely methanol, ethanol, acetone, and distilled water, for S. purpurea extract preparations. The root extract of S. purpurea, obtained by 100% acetone (S. purpurea-root-acetone), had the highest anticancer activities, antioxidation capacity (the DPPH activity with IC50 of 89.3 ± 2.2 μg/mL), antibacterial activities, total phenolic content (33.4 ± 0.7 mg GAE/g), and total flavonoid content (107.9 ± 2.2 mg QUE/g). The most abundant compounds in S. purpurea-root-acetone were identified using gas chromatography–mass spectrometry; 7,8-Dihydro-α-ionone was the major compound present in S. purpurea-root-acetone. In addition, the co-cytotoxicity of S. purpurea-root-acetone (combined with the clinical anticancer drug 5-fluorouracil (5-FU) on the survival, apoptosis, proliferation, and migration of the 4T1 mammary carcinoma) was examined. The combination of 5-FU with S. purpurea-root-acetone could be highly efficient for anti-4T1 cells. We also found that S. purpurea-root-acetone could inhibit the enzymatic activity of human dihydroorotase (huDHOase), an attractive target for potential anticancer chemotherapy. The sic most abundant compounds in S. purpurea-root-acetone were tested using an in silico analysis via MOE-Dock software for their binding affinities. The top-ranked docking conformations were observed for 7,8-dihydro-α-ionone and stigmast-5-en-3-ol, suggesting the inhibition potential against huDHOase. Overall, the collective data in this study may indicate the pharmacological potentials of S. purpurea-root-acetone for possible medical applications.

Stem cells work with their niches harmoniously during development. This concept has been extended to cancer pathology for cancer stem cells (CSCs) or cancer reprogramming. IGF-1R, a classical survival signaling, has been shown to regulate stem cell pluripotency, CSCs, or cancer reprogramming. The mechanism underlying such cell fate determination is unclear. We propose the determination is due to different niches in embryo development and tumor malignancy which modulate the consequences of IGF-1R signaling. Here we highlight the modulations of these niche parameters (hypoxia, inflammation, extracellular matrix), and the targeted stem cells (embryonic stem cells, germline stem cells, and mesenchymal stem cells) and CSCs, with relevance to cancer reprogramming. We organize known interaction between IGF-1R signaling and distinct niches in the double-sided cell fate with emerging trends highlighted. Based on these new insights, we propose that, through targeting IGF-1R signaling modulation, stem cell therapy and cancer stemness treatment can be further explored.

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play a central role in cells by participating in DNA metabolism, including replication, repair, recombination, and replication fork restart. SSBs are essential for cell survival and thus an attractive target for potential anti-pathogen chemotherapy. In this study, we determined the crystal structure and examined the size of the ssDNA-binding site of an SSB from Salmonella enterica serovar Typhimurium LT2 (SeSSB), a ubiquitous opportunistic pathogen which is highly resistant to antibiotics. The crystal structure was solved at a resolution of 2.8 Å (PDB ID 7F25), indicating that the SeSSB monomer possesses an oligonucleotide/oligosaccharide-binding (OB) fold domain at its N-terminus and a flexible tail at its C-terminus. The core of the OB-fold in the SeSSB is made of a six-stranded β-barrel capped by an α-helix. The crystal structure of the SeSSB contained two monomers per asymmetric unit, which may indicate the formation of a dimer. However, the gel-filtration chromatography analysis showed that the SeSSB forms a tetramer in solution. Through an electrophoretic mobility shift analysis, we characterized the stoichiometry of the SeSSB complexed with a series of ssDNA dA homopolymers, and the size of the ssDNA-binding site was determined to be around 22 nt. We also found the flavanonol taxifolin, also known as dihydroquercetin, capable of inhibiting the ssDNA-binding activity of the SeSSB. Thus, this result extended the SSB interactome to include taxifolin, a natural product with a wide range of promising pharmacological activities.

Endoplasmic reticulum (ER) stress mediates the effects of obesity on aggravating sepsis-induced lung injury. We investigated whether exosomes from human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) can mitigate pulmonary ER stress, lung injury, and the mechanisms of inflammation, oxidation, and apoptosis in lipopolysaccharide-treated obese mice. Diet-induced obese (DIO) mice (adult male C57BL/6J mice fed with a 12-week high-fat diet) received lipopolysaccharide (10 mg/kg, i.p.; DIOLPS group) or lipopolysaccharide plus exosomes (1 × 108 particles/mouse, i.p.; DIOLPSExo group). Our data demonstrated lower levels of ER stress (upregulation of glucose-regulated protein 78, phosphorylated eukaryotic initiation factor 2α, and C/EBP homologous protein; p = 0.038, p = 0.03, p < 0.001), and apoptosis (DNA fragmentation, p = 0.003) in lung tissues, as well as lower lung injury level (decreases in tidal volume, peak inspiratory flow, and end expiratory volume; increases in resistance, injury score, and tissue water content; p < 0.001,

Current guidelines recommend antibiotic prophylaxis for all patients with various degrees of cirrhosis and upper gastrointestinal (UGI) bleeding. This study assessed the need for antibiotic prophylaxis in patients with low Child-Pugh scores. We retrospectively screened all patients with cirrhosis who underwent upper endoscopies for UGI bleeding in a referral hospital in Taiwan between 2003 and 2014, from which 913 patients were enrolled after excluding patients with active bacterial infections, recent antibiotic use, early death, and Child-Pugh class C cirrhosis. Among them, 73 (8%) received prophylactic antibiotics, and 45 (4.9%) exhibited 14-day bacterial infection. Neither Child-Pugh score nor model for end stage liver disease score were optimal for predicting bacterial infection because their areas under the curves were 0.610 (95% confidence interval [CI]: 0.529-0.691) and 0.666 (95% CI: 0.591-0.742), respectively. Antibiotic prophylaxis did not reduce the risks of 14-day bacterial infection (relative risk [RR]: 0.932, 95% CI: 0.300-2.891, P = 0.902), 14-day rebleeding (RR: 0.791, 95% CI: 0.287-2.181, P = 0.650), or 42-day mortality (RR: 2.710, 95% CI: 0.769-9.524, P = 0.121). The results remained similar after propensity score adjustment. On-demand antibiotic treatment might suffice for patients with Child-Pugh class A/B cirrhosis and UGI bleeding.

Lymphoid enhancer factor 1 (LEF1) activity is associated with progression of several types of cancers. The role of LEF1 in progression of hepatocellular carcinoma (HCC) remains poorly known. We investigated LEF1 expression in HCC and its interactions with epithelial‐mesenchymal transition (EMT) regulators (e.g., Snail, Slug, Twist) and stemness genes (e.g., octamer‐binding transcription factor 4 [Oct4], sex determining region Y‐box 2 [Sox2], Nanog homeobox [Nanog]). Microarray analysis was performed on resected tumor samples from patients with HCC with or without postoperative recurrence. LEF1 expression was associated with postoperative recurrence as validated by immunohistochemical staining in another HCC cohort. Among 74 patients, 44 displayed a relatively high percentage of LEF1 staining (>30% of HCC cells), which was associated with a reduced recurrence‐free interval (P < 0.001) and overall survival (P = 0.009). In multivariate analysis, a high percentage of LEF1 staining was significantly associated with low albumin level (P = 0.035), Twist overexpression (P = 0.018), Snail overexpression (P = 0.064), co‐expression of Twist and Snail (P = 0.054), and multinodular tumors (P = 0.025). Down‐regulation of LEF1 by short hairpin RNA decreased tumor sphere formation, soft agar colony formation, and transwell invasiveness of HCC cell lines Mahlavu and PLC. Xenotransplant and tail vein injection experiments revealed that LEF1 down‐regulation in Mahlavu reduced tumor size and metastasis. LEF1 up‐regulation in Huh7 increased sphere formation, soft agar colony formation, and transwell invasiveness. LEF1 was shown to physically interact with and transcriptionally activate promoter regions of Oct4, Snail, Slug, and Twist. Furthermore, Oct4, Snail, and Twist transactivated LEF1 to form a regulatory positive‐feedback loop. Conclusion: LEF1 plays a pivotal role in HCC progression through transcriptional regulation of Oct4 and EMT regulators.

Summary: Hypoxia cooperates with endocrine signaling to maintain the symmetric self-renewal proliferation and migration of embryonic germline stem cells (GSCs). However, the lack of an appropriate in vitro cell model has dramatically hindered the understanding of the mechanism underlying this cooperation. Here, using a serum-free system, we demonstrated that hypoxia significantly induced the GSC mesenchymal transition, increased the expression levels of the pluripotent transcription factor OCT4 and migration-associated proteins (SDF-1, CXCR4, IGF-1, and IGF-1R), and activated the cellular expression and translocalization of the CXCR4-downstream proteins ARP3/pFAK. The underlying mechanism involved significant IGF-1/IGF-1R activation of OCT4/CXCR4 expression through HIF-2α regulation. Picropodophyllin-induced inhibition of IGF-1R phosphorylation significantly suppressed hypoxia-induced SDF-1/CXCR4 expression and cell migration. Furthermore, transactivation between IGF-1R and CXCR4 was involved. In summary, we demonstrated that niche hypoxia synergistically cooperates with its associated IGF-1R signaling to regulate the symmetric division (self-renewal proliferation) and cell migration of alkaline phosphatase-positive GSCs through HIF-2α-OCT4/CXCR4 during embryogenesis. : In this article, Huang and colleagues demonstrate that niche hypoxia promotes symmetric self-renewal proliferation and migration of PGC-like CD49f+AP+GSCs through IGF-IR regulation. Using a serum-free culture system, the crosstalk between IGF-1R and CXCR4 signaling was discovered. This work demonstrated that embryonic hypoxia synergistically cooperated with IGF-1R signaling to regulate the symmetric self-renewal and migration of PGC-like GSCs through a HIF-2α–OCT4/CXCR4 loop. Keywords: hypoxia, niche, germline stem cells, self-renewal, migration, IGF-1R, HIF-2α, OCT4, SDF-1, CXCR4

Obesity complicates sepsis and increases the mortality of sepsis. We examined the effects of exosomes (from human placenta choriodecidual membrane-derived mesenchymal stem cells, pcMSCs) on preventing sepsis in obesity and the mitigating role of hsa-let-7i-5p microRNA. Obese mice (adult male C57BL/6J mice fed a high-fat diet for 12 weeks) received normal saline (HFD), endotoxin (10 mg/kg, intraperitoneal (ip); HFDLPS), endotoxin with exosomes (1 × 108 particles/mouse, ip; HLE), or endotoxin with let-7i-5p microRNA inhibitor-pretreated exosomes (1 × 108 particles/mouse, ip; HLEi). Our data demonstrated that the 48-h survival rate in the HLE (100%) group was significantly higher than in the HFDLPS (50%) and HLEi (58.3%) groups (both p < 0.05). In the surviving mice, by contrast, levels of liver injury (injury score, plasma aspartate transaminase and alanine transaminase concentrations, tissue water content, and leukocyte infiltration in liver tissues; all p < 0.05), inflammation (nuclear factor-κB activation, hypoxia-inducible factor-1α activation, macrophage activation, and concentrations of tumor necrosis factor-α, interleukin-6, and leptin in liver tissues; all p < 0.05), and oxidation (malondialdehyde in liver tissues, with p < 0.001) in the HLE group were significantly lower than in the HFDLPS group. Levels of mitochondrial injury/dysfunction and apoptosis in liver tissues in the HLE group were also significantly lower than in the HFDLPS group (all p < 0.05). Inhibition of let-7i-5p microRNA offset the effects of the exosomes, with most of the aforementioned measurements in the HLEi group being significantly higher than in the HLE group (all p < 0.05). In conclusion, exosomes mitigated endotoxin-induced mortality and liver injury in obese mice, and these effects were mediated by let-7i-5p microRNA.

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.

This study evaluated the biocompatibility and biological performance of novel additive-manufactured bioabsorbable iron-based porous suture anchors (iron_SAs). Two types of bioabsorbable iron_SAs, with double- and triple-helical structures (iron_SA_2_helix and iron_SA_3_helix, respectively), were compared with the synthetic polymer-based bioabsorbable suture anchor (polymer_SAs). An in vitro mechanical test, MTT assay, and scanning electron microscope (SEM) analysis were performed. An in vivo animal study was also performed. The three types of suture anchors were randomly implanted in the outer cortex of the lateral femoral condyle. The ultimate in vitro pullout strength of the iron_SA_3_helix group was significantly higher than the iron_SA_2_helix and polymer_SA groups. The MTT assay findings demonstrated no significant cytotoxicity, and the SEM analysis showed cells attachment on implant surface. The ultimate failure load of the iron_SA_3_helix group was significantly higher than that of the polymer_SA group. The micro-CT analysis indicated the iron_SA_3_helix group showed a higher bone volume fraction (BV/TV) after surgery. Moreover, both iron SAs underwent degradation with time. Iron_SAs with triple-helical threads and a porous structure demonstrated better mechanical strength and high biocompatibility after short-term implantation. The combined advantages of the mechanical superiority of the iron metal and the possibility of absorption after implantation make the iron_SA a suitable candidate for further development.

Hereditary anemia has various manifestations, such as sickle cell disease (SCD), Fanconi anemia, glucose-6-phosphate dehydrogenase deficiency (G6PDD), and thalassemia. The available management strategies for these disorders are still unsatisfactory and do not eliminate the main causes. As genetic aberrations are the main causes of all forms of hereditary anemia, the optimal approach involves repairing the defective gene, possibly through the transplantation of normal hematopoietic stem cells (HSCs) from a normal matching donor or through gene therapy approaches (either in vivo or ex vivo) to correct the patient’s HSCs. To clearly illustrate the importance of cell and gene therapy in hereditary anemia, this paper provides a review of the genetic aberration, epidemiology, clinical features, current management, and cell and gene therapy endeavors related to SCD, thalassemia, Fanconi anemia, and G6PDD. Moreover, we expound the future research direction of HSC derivation from induced pluripotent stem cells (iPSCs), strategies to edit HSCs, gene therapy risk mitigation, and their clinical perspectives. In conclusion, gene-corrected hematopoietic stem cell transplantation has promising outcomes for SCD, Fanconi anemia, and thalassemia, and it may overcome the limitation of the source of allogenic bone marrow transplantation.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC–MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

In the original publication of this article [1], labelling within Fig. 7a was incorrect. The updated figure is shown below, with ‘DMT1’ now corrected to read ‘DNMT1’.

Insulin-like Growth Factor (IGF)/IGF-1 Receptor (IGF-1R) signaling is known to regulate stem cell pluripotency and differentiation to trigger cell proliferation, organ development, and tissue regeneration during embryonic development. Unbalanced IGF/IGF-1R signaling can promote cancer cell proliferation and activate cancer reprogramming in tumor tissues, especially in the liver. Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, with a high incidence and mortality rate in Asia. Most patients with advanced HCC develop tyrosine kinase inhibitor (TKI)-refractoriness after receiving TKI treatment. Dysregulation of IGF/IGF-1R signaling in HCC may activate expression of cancer stemness that leads to TKI refractoriness and tumor recurrence. In this review, we summarize the evidence for dysregulated IGF/IGF-1R signaling especially in hepatitis B virus (HBV)-associated HCC. The regulation of cancer stemness expression and drug resistance will be highlighted. Current clinical treatments and potential therapies targeting IGF/IGF-1R signaling for the treatment of HCC will be discussed.

The use of immune checkpoint inhibitors (ICI) in treating cancer has revolutionized the approach to eradicate cancer cells by reactivating immune responses. However, only a subset of patients benefits from this treatment; the majority remains unresponsive or develops resistance to ICI therapy. Increasing evidence suggests that metabolic machinery in the tumor microenvironment (TME) plays a role in the development of ICI resistance. Within the TME, nutrients and oxygen are scarce, forcing immune cells to undergo metabolic reprogramming to adapt to harsh conditions. Cancer-induced metabolic deregulation in immune cells can attenuate their anti-cancer properties, but can also increase their immunosuppressive properties. Therefore, targeting metabolic pathways of immune cells in the TME may strengthen the efficacy of ICIs and prevent ICI resistance. In this review, we discuss the interactions of immune cells and metabolic alterations in the TME. We also discuss current therapies targeting cellular metabolism in combination with ICIs for the treatment of cancer, and provide possible mechanisms behind the cellular metabolic rewiring that may improve clinical outcomes.

Adjuvant 5-fluorouracil (5-FU)-based chemotherapy, including FOLFOX (5-FU, leucovorin, and oxaliplatin), is recommended for colorectal cancer. However, intestinal mucositis remains a common adverse effect for which no effective preventive strategies are available. To develop a convenient and novel way to alleviate mucositis, we investigated the effect of Lactobacillus casei variety rhamnosus (Lcr35) on FOLFOX-induced mucosal injury. BALB/c mice subcutaneously injected with syngeneic CT26 colorectal adenocarcinoma cells were orally administered Lcr35 daily before, during, and after 5-day injection of FOLFOX regimen, for 14 days. The following methods were used: diarrhea score for toxicity, ELISA for cytokine production, histopathology for intestinal injury, immunohistochemistry for apoptosis/proliferation and regulatory proteins, RT-PCR for cytokine mRNA expression, and DNA sequencing for fecal gut microbiota. FOLFOX administration to colorectal cancer-bearing mice significantly inhibited tumor growth and the accompanying marked diarrhea and intestinal injury histologically characterized by the shortening of villi and destruction of intestinal crypts. Preventive administration of Lcr35 dose-dependently reduced the severity of diarrhea and intestinal mucositis without affecting the anti-tumor effect of FOLFOX. The numbers of apoptotic, NF-κB-, and BAX-activated cells increased after FOLFOX, and these responses were mitigated by Lcr35. TNF-α and IL-6 upregulation by FOLFOX treatment was attenuated by Lcr35. The fecal gut microbiota composition of Firmicutes and Bacteroidetes disturbed by FOLFOX was significantly reversed by Lcr35 toward a preferential profile. In conclusion, the oral probiotic Lcr35 prevented FOLFOX-induced intestinal mucositis in colorectal cancer-bearing mice. The putative mechanism might involve modulation of gut microbiota and proinflammatory responses with suppression of intrinsic apoptosis in intestinal injury.

Dihydropyrimidinase, a dimetalloenzyme containing a carboxylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. Unlike all known dihydropyrimidinases, which are tetrameric, pseudomonal dihydropyrimidinase forms a dimer at neutral pH. In this paper, we report the crystal structure of P. aeruginosa dihydropyrimidinase at pH 5.9 (PDB entry 5YKD). The crystals of P. aeruginosa dihydropyrimidinase belonged to space group C2221 with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 Å. The structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å resolution. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa dihydropyrimidinase monomers. Gel filtration chromatographic analysis of purified P. aeruginosa dihydropyrimidinase revealed a mixture of dimers and tetramers at pH 5.9. Thus, P. aeruginosa dihydropyrimidinase can form a stable tetramer both in the crystalline state and in the solution. Based on sequence analysis and structural comparison of the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, different oligomerization mechanisms are proposed.