Experimental Biology and Medicine

COVID-19 or SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome/pneumonia with features of cytokine storm reminiscent of secondary hemophagocytic lymphohistiocytosis (HLH), which can be diagnosed by the calculated HScore. Recent reports have suggested favorable responses to the interleukin-1 receptor antagonist, anakinra in patients with COVID-19 associated secondary HLH. In our single institution study, we compared 14 COVID-19 cytokine storm patients with 10 secondary HLH patients seen immediately prior to the pandemic (non-COVID-19), to determine whether diagnostic features of secondary HLH were typically seen in COVID-19 patients presenting with cytokine storm. Although most of our COVID-19 patients did not fulfill diagnostic criteria for HLH, we hypothesize that identification of HLH may relate to the severity or timing of cytokine release. Based on our observations, we would suggest distinguishing between cytokine release syndrome and secondary HLH, reserving the latter term for cases fulfilling diagnostic criteria.

Nearly 60% of Plasmodium falciparum proteins are still uncharacterized and their functions are unknown. In this report, we carried out the functional characterization of a 45 kDa protein (PF3D7_1459400) and showed its potential as a target for blood stage malaria vaccine development. Analysis of protein subcellular localization, native protein expression profile, and erythrocyte invasion inhibition of both clinical and laboratory parasite strains by peptide antibodies suggest a functional role of PF3D7_1459400 protein during erythrocyte invasion. Also, immunoreactivity screens using synthetic peptides of the protein showed that adults resident in malaria endemic regions in Ghana have naturally acquired plasma antibodies against PF3D7_1459400 protein. Altogether, this study presents PF3D7_1459400 protein as a potential target for the development of peptide-based vaccine for blood-stage malaria.

As a flavonoid, baicalein exhibits remarkable anti-cancer roles in several cancers. However, the factors regulating the antitumorigenic roles of baicalein in cervical cancer remain undefined. Here, we revealed that long noncoding RNA SNHG1 is implicated in the tumor-suppressive roles of baicalein. Functional assays demonstrated that ectopic expression of SNHG1 attenuates the roles of baicalein in repressing cervical cancer cell viability, inducing apoptosis, and repressing migration. SNHG1 silencing promotes the tumor-suppressive roles of baicalein in cervical cancer cell viability, apoptosis, and migration. Xenograft assays showed that SNHG1 reverses the tumor-suppressive roles of baicalein in repressing cervical cancer growth in vivo. Mechanistic investigations revealed that SNHG1 directly binds miR-3127-5p and up-regulates FZD4, a target of miR-3127-5p. Via regulating miR-3127-5p/FZD4, SNHG1 activates Wnt/β-catenin signaling. Moreover, SNHG1 reverses the repressive role of baicalein on Wnt/β-catenin signaling. The effect of SNHG1 on the antitumorigenic process of baicalein was abolished by Wnt/β-catenin signaling inhibitor ICG-001. Together, our observations demonstrated that SNHG1 represses the tumor-suppressive roles of baicalein in cervical cancer through regulating miR-3127-5p/FZD4/Wnt/β-catenin axis, and suggested that targeting SNHG1 represents a potential strategy to enhance the tumor-suppressive roles of baicalein in cervical cancer.

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer.

Several mutations act as driver mutations in breast cancer, including GATA3 mutations. Reports of the relation between GATA3 mutations and breast cancer prognosis remain conflicting. Also, the role of GATA3 germline mutations is not well studied. We hypothesize that different mutation types could have different effects. Also, this study aims to assess effect of GATA3 mutations on GATA3 protein function as a transcription factor, and target pathways affected. DNA from de novo breast cancer female patients was sequenced to detect exon 6 GATA3 mutation. Sequence analysis was performed along with clinical and prognostic parameters and disease-free survival. Public datasets were analyzed for differentially expressed genes and pathways with mutant GATA3 patients. Mutations in GATA3 exon 6 were detected in 56.1% of patients (including 2 novel, Lys368fs, Pro354Lys). Intronic mutations were significantly higher in long disease-free survival group, while frameshift mutations were significantly higher in short DFS group. Patients with tumor size ≥20 had significantly higher protein coding and lower intronic mutations compared to patients with tumor size <20 mm. Differential expression and pathway analysis showed that mutant GATA3 had lost its negative regulatory effect on several pathways such as: signaling by interleukins, regulation of TP53 expression, and RUNX3 regulated CDKN1A transcription pathway. PIK3CA, SKP1, FBP1, SMAD3, ANXA9 and CLSTN2 were positively correlated to wild-type GATA3 expression, but not mutant GATA3. Intronic germline mutations of GATA3 could be related to better prognosis, while protein coding GATA3 germline mutations could be related to unfavorable prognosis. GATA3 mutations lead to dysregulation of pathways related to immunity, breast cancer development, and metabolism.

In the last decade, the inclusion of HPV DNA testing in cervical cancer screening has provided one of the best strategies for the prevention and timely detection of HPV. We conducted a high-throughput HPV genotyping study based on MALDI-TOF mass spectrometry to determine the prevalence of 24 HPV genotypes, including oncogenic genotypes, in Mexican women and correlated the results with cytological findings and clinical variables. We likewise identified the risk factors in patients with the HPV infection. Our study included 1000 women from Sonora, Mexico, who participated in cervical cancer screening campaigns and who underwent a Pap smear and HPV DNA test. The results showed that the overall prevalence of HPV was 27.2%, 18.5% with single, and 8.7% multiple infections. The low-risk HPV genotype 6 (8.5%) and oncogenic genotypes 31 (8.1%) and 53 (4.4%) were the most prevalent in the study population. The number of lifetime sexual partners, previous STIs, and age at first intercourse was significantly associated with HPV infection (P ≤ 0.05). Smoking (OR = 1.5609; 95% IC 1.062–2.292) and more than three lifetime sexual partners (OR = 1.609; 95% IC = 1.124–2.303) represented risk factors for HPV infection. Cytological abnormalities were found in 3.4% of the HPV-positive samples. CIN 1–3 occurred in 0.6% of high-risk HPV cases. In general, the prevalence of the HPV genotypes is high in Mexican women with normal cytological findings. This issue highlights the importance of HPV research in seemingly healthy women and could help guide screening strategies for cervical cancer prevention in Mexico.

Interleukin (IL)-17A, a proinflammatory cytokine produced by T-helper (Th)17 cells, has been associated with autoimmune diseases. Type 1 diabetes (T1D) is caused either due to mutation of insulin gene or developed as an autoimmune disease. Studies have shown that IL-17A expression is upregulated in the pancreas in T1D patients and animal models. However, role or importance of IL-17A in T1D pathogenesis needs elucidation. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells through activating IL-17 receptor A (IL-17RA) is lacking. Ins2^Akita (Akita) mouse, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis, was crossed with IL-17A-knockout mouse and male IL-17A-deficient Akita mice were used. Streptozotocin, a pancreatic β-cell-specific cytotoxin, was employed to induce a diabetic model in MIN6 cells, a mouse insulinoma cell line. IL-17A expression in the pancreas was upregulated in both Akita and streptozotocin-induced diabetic mice. IL-17A-knockout Akita mice manifested reduced blood glucose concentration and raised serum insulin level. IL-17A deficiency also decreased production of the proinflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, and interferon (IFN)-γ in Akita mice. IL-17RA expression in MIN6 cells was upregulated by IL-17A. IL-17A enhanced expression of TNF-α, IL-1β, IFN-γ, and inducible nitric oxide synthase (iNOS) and further increased streptozotocin-induced expression of the inflammatory factors in MIN6 cells. IL-17A exacerbated streptozotocin-induced MIN6 cell apoptosis and insulin secretion impairment. Blocking IL-17RA with anti-IL-17RA-neutralizing antibody reduced all these deleterious effects of IL-17A on MIN6 cells. Collectively, IL-17A deficiency alleviated hyperglycemia, hypoinsulinemia, and inflammatory response in Akita mice that are characteristic for T1D. IL-17A exerted an alone and synergistic destruction with streptozotocin to pancreatic β cells through IL-17RA pathway. Thus, the data suggest that targeting IL-17A and/or IL-17RA is likely to preserve remaining β-cell function and treat T1D.

High fat intake is one of the most important reasons of the surging prevalence of childhood obesity all over the world. Obesity and high fat intake have been revealed to cause premature activation of hypothalamo-pituitary-gonadal axis and central precocious puberty. The onset of puberty is controlled by neuroendocrine mechanisms containing overlapping and interacting gene networks. The latter contains five major transcriptional level hubs, among which the transcriptional factor p53, a well-established tumor suppressor protein, also plays a crucial role in obesity and metabolic disorders. In the current study, we repeated prior observations that high-fat diet advances vaginal opening in rodents and extended these findings by demonstrating that high-fat diet mice had higher expression of p53 in hypothalami than mice fed with normal chow. More importantly, in high-fat diet mice, hypothalamus-specific overexpression of p53 can make vaginal opening much earlier, while inhibition of p53 expression relatively delayed vaginal opening. The c-Myc and Lin28b levels increased, while let-7a mRNA levels decreased in the high-fat diet mice. Overexpression of p53 reduced c-Myc and Lin28b mRNA and protein levels, whereas elevated let-7a mRNA levels in high-fat diet mice. Inhibition of p53 expression by pifithrin-α elevated c-Myc and Lin28b but reduced let-7a levels in high-fat diet mice. In conclusion, high fat intake can accelerate the onset of puberty by up-regulation of p53 expression in hypothalamus. Overexpressed p53 may accelerate hypothalamo-pituitary-gonadal axis activation partially through the c-Myc/Lin28/let-7 system.

Nasopharyngeal cancer is one of the most common malignant tumors in the head and neck. Identification of promising miRNA biomarkers might benefit a lot to the detection of nasopharyngeal carcinoma. miRNA expression profile and clinical information were obtained from two microarray profiling data sets from the Gene Expression Omnibus (GEO) database. miRNA signature model was constructed via univariate Cox survival analysis, multivariate Cox survival analysis, and least absolute shrinkage and selection operator Cox regression analysis. Kaplan–Meier curve, area under the curve (AUC), decision curve analysis, Box plot, and nomogram were used to evaluate the prognosis of the model to patients. 67 up-regulated and 93 down-regulated miRNAs were identified from GEO microarray data sets (P < 0.05). A three-miRNA signature (has-miR-142-3p, has-miR-29c, and has-miR-30e) was obviously associated with the overall survival of nasopharyngeal carcinoma patients (P  < 0.001). The AUCs for the signature were 0.74, 0.7 for the training set and external validation set. The AUC of disease free survival and distant metastasis-free survival were also high. The model has better clinical independence and has better clinical prediction effect when combined with clinical characteristics (P < 0.0001). Compared with the published models, our model had a higher AUC. Our results revealed that a three-miRNA signature was a potential novel prognostic biomarker for nasopharyngeal carcinoma.

Acute myeloid leukemia (AML) is a malignant clonal disease derived from hematopoietic stem/progenitor cell. Leukemia blasts cause extensive hypoxia of bone marrow (BM), which lead to disorder and remodeling of BM niche, thereby becoming “leukemic niche” to support the development and drug-resistance of AML as well as the maintenance of normal hematopoietic stem cells. In this study, the biological characteristics (such as self-renewal, apoptosis, migration, autocrine) and function (vascularization) of mesenchymal stem cells (MSCs) and human umbilical artery endothelial cells (HUAECs) that make up BM arteriolar niche in simulated hypoxia AML context were investigated. It was found that moderate hypoxia enhanced the viability of the arteriolar niche cells, but severe hypoxia of AML BM resulted in the damage of arteriolar niche cells and the disorder of vascular cytokines C-X-C motif chemokine ligand 6 (CXCL6). The dynamic changes of CXCL6 in the system as well as its anti-apoptotic and promoting angiogenic effects suggested that CXCL6 played an important role in the remodeling of BM arteriolar niche in AML. Taking advantage of CXCL6 can save the damaged MSCs and HUAECs, which is the hope of rescuing arteriolar niche. It is suggested that CXCL6 may be an assistant strategy for microenvironment targeted therapy of AML.

Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.

This study aims to determine whether sickle cell mice could recapitulate features of cognitive and neurobehavioral impairment observed in sickle cell patients and whether neuroinflammation could be a potential therapeutic target as in other non-sickle cell disease-related cognitive dysfunction. Cognitive (learning and memory) and behavioral (anxiety) deficits in 13- and later 6-month-old male Townes humanized sickle cell (SS) and matched control (AA) mice were evaluated using novel object recognition (NOR) and fear conditioning tests. Immunohistochemistry was performed to quantify peripheral immune cell (CD45⁺) and activated microglia (Iba1⁺) as markers of neuroinflammation in the dentate and peri-dentate gyrus areas. We evaluated cell fate by measuring 5'-bromodeoxyuridine and doublecortin fluorescence and phenotyped proliferating cells using either glial fibrillary acid protein (GFAP⁺), neuronal nuclei (NeuN⁺), CD45⁺, and Iba1⁺. In addition, Golgi-Cox staining was used to assess markers of neuroplasticity (dendritic spine density and morphology and density of dendrite arbors) on cortical and hippocampal pyramidal neurons. Compared to matched AA controls, 13-month-old SS mice showed significant evidence of cognitive and behavioral deficit on NOR and fear conditioning tests. Also, SS mice had significantly higher density of CD45⁺ and activated microglia cells (i.e. more evidence of neuroinflammation) in the dentate and peri-dentate gyrus area. Additionally, SS mice had significantly lower dendritic spine density, but a higher proportion of immature dendritic spines. Treatment of 13-month-old SS mice with minocycline resulted in improvement of cognitive and behavioral deficit compared to matched vehicle-treated SS mice. Also, treated SS mice had significantly fewer CD45⁺ and activated microglia cells (i.e. less evidence of neuroinflammation) in the dentate and peri-dentate gyrus, as well as a significant improvement in markers of neuroplasticity.