The aim of this study was to test the hypothesis that hepatic vitamin C (VC) levels in VC deficient mice rescued with high doses of VC supplements still do not reach the optimal levels present in wild-type mice. For this, we used a mouse scurvy model (sfx) in which the L-gulonolactone oxidase gene (Gulo) is deleted. Six age- (6 weeks old) and gender- (female) matched wild-type (WT) and sfx mice (rescued by administering 500 mg of VC/L) were used as the control (WT) and treatment (MT) groups (n = 3 for each group), respectively. Total hepatic RNA was used in triplicate microarray assays for each group. EDGE software was used to identify differentially expressed genes and transcriptomic analysis was used to assess the potential genetic regulation of Gulo gene expression. Hepatic VC concentrations in MT mice were significantly lower than in WT mice, even though there were no morphological differences between the two groups. In MT mice, 269 differentially expressed transcripts were detected (≥ twice the difference between MT and WT mice), including 107 up-regulated and 162 down-regulated genes. These differentially expressed genes included stress-related and exclusively/predominantly hepatocyte genes. Transcriptomic analysis identified a major locus on chromosome 18 that regulates Gulo expression. Since three relevant oxidative genes are located within the critical region of this locus we suspect that they are involved in the down-regulation of oxidative activity in sfx mice.

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

Dlx3 (distal-less homeobox 3) haploinsufficiency in mice has been shown to result in restricted fetal growth and placental defects. We previously showed that maternal choline supplementation (4X versus 1X choline) in the Dlx3+/- mouse increased fetal and placental growth in mid-gestation. The current study sought to test the hypothesis that prenatal choline would modulate indicators of placenta function and development. Pregnant Dlx3+/- mice consuming 1X (control), 2X, or 4X choline from conception were sacrificed at embryonic (E) days E10.5, E12.5, E15.5, and E18.5, and placentas and embryos were harvested. Data were analyzed separately for each gestational day controlling for litter size, fetal genotype (except for models including only +/- pups), and fetal sex (except when data were stratified by this variable). 4X choline tended to increase (p < 0.1) placental labyrinth size at E10.5 and decrease (p < 0.05) placental apoptosis at E12.5. Choline supplementation decreased (p < 0.05) expression of pro-angiogenic genes Eng (E10.5, E12.5, and E15.5), and Vegf (E12.5, E15.5); and pro-inflammatory genes Il1b (at E15.5 and 18.5), Tnfα (at E12.5) and Nfκb (at E15.5) in a fetal sex-dependent manner. These findings provide support for a modulatory effect of maternal choline supplementation on biomarkers of placental function and development in a mouse model of placental insufficiency.

Distant metastasis is the primary barrier for the successful treatment of patients with colorectal cancer, and thus, searching for new therapeutic targets by further exploring the molecular mechanisms of colorectal cancer metastasis is important. In this study, we investigated the biological and clinical significance of RASSF6 in colorectal cancer as well as the underlying molecular mechanisms. We found that low RASSF6 expression corresponds to a poor prognosis in colorectal cancer patients, and low RASSF6 expression is distinctly associated with tumour progression. Our in vitro analysis revealed that RASSF6 suppresses the proliferation and metastasis of DLD1 cells, and RASSF6 knockdown in HCT116 cells confirmed these observations. Our mechanistic investigation revealed that RASSF6 inhibits the expression of the classical target genes of Wnt signalling, as demonstrated by the reduced expression of TCF1, c-Jun, and c-Myc in RASSF6-overexpressing DLD1 stable cell lines. Furthermore, we show that RASSF6 functions as a negative regulator of the epithelial-mesenchymal transition; the expression levels of the epithelial markers ZO-1 and E-cadherin were increased, while the expression level of the mesenchymal marker Snail was decreased in a RASSF6-overexpressing DLD1 cell line. Additionally, rescue assays revealed that the activation of Wnt signalling by LiCl treatment impaired the inhibitory effect of RASSF6 on the proliferation and metastasis of colorectal cancer cells, which implies that RASSF6 suppresses the tumorigenicity of colorectal cancer cells at least in part through inhibiting Wnt signalling pathway. Collectively, these findings provide new perspectives for the future study of RASSF6 as a therapeutic target for colorectal cancer.

Environmental chemicals exposure is one of the primary factors for liver toxicity and hepatocarcinoma. Thioacetamide (TAA) is a well-known hepatotoxicant and could be a liver carcinogen in humans. The discovery of early and sensitive microRNA (miRNA) biomarkers in liver injury and tumor progression could improve cancer diagnosis, prognosis, and management. To study this, we performed next generation sequencing of the livers of Sprague-Dawley rats treated with TAA at three doses (4.5, 15 and 45 mg/kg) and four time points (3-, 7-, 14- and 28-days). Overall, 330 unique differentially expressed miRNAs (DEMs) were identified in the entire TAA-treatment course. Of these, 129 DEMs were found significantly enriched for the "liver cancer" annotation. These results were further complemented by pathway analysis (Molecular Mechanisms of Cancer, p53-, TGF-β-, MAPK- and Wnt-signaling). Two miRNAs (rno-miR-34a-5p and rno-miR-455-3p) out of 48 overlapping DEMs were identified to be early and sensitive biomarkers for TAA-induced hepatocarcinogenicity. We have shown significant regulatory associations between DEMs and TAA-induced liver carcinogenesis at an earlier stage than histopathological features. Most importantly, miR-34a-5p is the most suitable early and sensitive biomarker for TAA-induced hepatocarcinogenesis due to its consistent elevation during the entire treatment course.

Prostate-specific membrane antigen (PSMA) is expressed at high levels on malignant prostate cells and is likely an important therapeutic target for the treatment of prostate carcinoma. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines as well as passive administration of PSMA-specific monoclonal antibodies (mAb). Conventional mAb immunotherapy has numerous logistical and practical limitations, including high production costs and a requirement for frequent dosing due to short mAb serum half-life. In this report, we describe a novel strategy of antibody-based immunotherapy against prostate carcinoma that utilizes synthetic DNA plasmids that encode a therapeutic human mAb that target PSMA. Electroporation-enhanced intramuscular injection of the DNA-encoded mAb (DMAb) plasmid into mice led to the production of functional and durable levels of the anti-PSMA antibody. The anti-PSMA produced in vivo controlled tumor growth and prolonged survival in a mouse model. This is likely mediated by antibody-dependent cellular cytotoxicity (ADCC) effect with the aid of NK cells. Further study of  this novel approach for treatment of human prostate disease and other malignant conditions is warranted.

Vitamin C (VC) is well known as an antioxidant in humans, primates and guinea pigs. Studies have suggested gender differences in VC requirements in humans, and gender differences in oxidant injury vulnerability in early life may represent a biological mechanism contributing to gender disparity in later life. Using spontaneous bone fracture (sfx) mice, which lack the gene for L-Gulonolactone oxidase (Gulo), we studied the potential sex difference in expression profiles of oxidative genes at the whole-genome level. Then, we analyzed data of gene expressions in a mouse population of recombinant inbred (RI) strains originally derived by crossing C57BL/6J (B6) and DBA/2J (D2) mice. Our data indicated that there were sex differences in the regulation of pre- and pro-oxidative genes in sfx mice. The associations of expression levels among Gulo, its partner genes and oxidative genes in the BXD (B6 × D2) RI strains showed a sex difference. Transcriptome mapping suggests that Gulo was regulated differently between female and male mice in BXD RI strains. Our study indicates the importance of investigating sex differences in Gulo and its oxidative function by using available mouse models.

Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

The placental epigenome regulates processes that affect placental and fetal development, and could be mediating some of the reported effects of maternal choline supplementation (MCS) on placental vascular development and nutrient delivery. As an extension of work previously conducted in pregnant mice, the current study sought to explore the effects of MCS on various epigenetic markers in the placenta. RNA and DNA were extracted from placentas collected on embryonic day 15.5 from pregnant mice fed a 1X or 4X choline diet, and were subjected to genome-wide sequencing procedures or mass-spectrometry-based assays to examine placental imprinted gene expression, DNA methylation patterns, and microRNA (miRNA) abundance. MCS yielded a higher (fold change = 1.63-2.25) expression of four imprinted genes (Ampd3, Tfpi2, Gatm and Aqp1) in the female placentas and a lower (fold change = 0.46-0.62) expression of three imprinted genes (Dcn, Qpct and Tnfrsf23) in the male placentas (false discovery rate (FDR) ≤ 0.05 for both sexes). Methylation in the promoter regions of these genes and global placental DNA methylation were also affected (p ≤ 0.05). Additionally, a lower (fold change = 0.3; Punadjusted = 2.05 × 10-4; FDR = 0.13) abundance of miR-2137 and a higher (fold change = 1.25-3.92; p < 0.05) expression of its target genes were detected in the 4X choline placentas. These data demonstrate that the placental epigenome is responsive to maternal choline intake during murine pregnancy and likely mediates some of the previously described choline-induced effects on placental and fetal outcomes.

Influenza virus remains a significant public health threat despite innovative vaccines and antiviral drugs. A major limitation to current vaccinations and therapies against influenza virus is pathogenic diversity generated by shift and drift. A simple, cost-effective passive immunization strategy via in vivo production of cross-protective antibody molecules may augment existing vaccines and antiviral drugs in seasonal and pandemic outbreaks. We engineered synthetic plasmid DNA to encode two novel and broadly cross-protective monoclonal antibodies targeting influenza A and B. We utilized enhanced in vivo delivery of these plasmid DNA-encoded monoclonal antibody (DMAb) constructs and show that this strategy induces robust levels of functional antibodies directed against influenza A and B viruses in mouse sera. Mice receiving a single inoculation with anti-influenza A DMAb survive lethal Group 1 H1 and Group 2 H3 influenza A challenges, while inoculation with anti-influenza B DMAb yields protection against lethal Victoria and Yamagata lineage influenza B morbidity and mortality. Furthermore, these two DMAbs can be delivered coordinately resulting in exceptionally broad protection against both influenza A and B. We demonstrate this protection is similar to that achieved by conventional protein antibody delivery. DMAbs warrant further investigation as a novel immune therapy platform with distinct advantages for sustained immunoprophylaxis against influenza.

Mangosteen (Garcinia mangostana L.) is one of the most popular tropical fruits (called the "Queen of Fruits"), and is a rich source of oxygenated and prenylated xanthone derivatives. In the present work, phytochemical investigation has resulted in one new prenylated xanthone and 13 known xanthones isolated from the pericarp of G. mangostana. Their structures were established by spectroscopic data analysis, including X-ray diffraction. The new one was further tested for cytotoxic activity against seven cancer cell lines (CNE-1, CNE-2, A549, H490, PC-3, SGC-7901, U87), displaying the half maximal inhibitory concentration (IC₅0) values 3.35, 4.01, 4.84, 7.84, 6.21, 8.09, and 6.39 μM, respectively. It is noteworthy that the new compound can promote CNE-2 cells apoptosis in late stage, having a remarkable inhibition effect on the side population growth of CNE-2 at 1.26 μM. The bioactive compound was also detected in extract from fresh mangosteen flesh, which indicated that the popular fruit could have potential cytotoxic activity for cancer cell lines.

Qigong, as one of the essential elements of Traditional Chinese exercises, has been used to improve physical and psychological health and combat diseases in China for thousands of years. In recent years, the beneficial effects of Qigong on different medical conditions are becoming more accepted by both patients and health care providers. Although it is a common impression that Qigong and related therapies are generally safe procedures, but the current understanding of its adverse events is fragmented. Thus, we conducted this overview to synthesize comprehensively existing systematic reviews on adverse events associated with Qigong and related therapies, and our findings can be used to informing clinicians, Qigong practitioner, and patients alike on applying such treatments or interventions in clinical treatment and daily life training mindful manner, and provide a guideline for researchers in future.

Tankyrase1 (TNKS1), which often shows abnormal expression in many malignant tumor cells, plays an important role in tumor progression. In our previous study, we found that TNKS1 is also closely related to pathologic grade in human astrocytoma and its expression level is positively correlated with the Wnt/β-catenin pathway. This study is aimed to further elucidate the biological functions of TNKS1 as well as its relationship with the Wnt/β-catenin pathway.

Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.

Depressive disorder has gradually become one of the most commonly reported disabling psychiatric complication that occurs after traumatic brain injury (TBI). Currently classical antidepressant medications may not have the same effectiveness in patients with TBI as in patients without TBI. Non-pharmacological interventions have been considered to be effective for managing depressive symptoms or treating depressive disorder. But to date the comparative effectiveness of various types of non-pharmacological interventions has been synthesized in few studies, the evidence remains inconclusive. Thus, the purpose of this systematic review and network meta-analyses is to summarize high-quality evidence and identify the most effective non-pharmacological intervention when applied to treat the depressive disorder in patients after TBI.

Glioma is the most common primary intracranial carcinoma with extremely poor prognosis. The significances of long non-coding RNA (lncRNA) involved in glioma have been started revealed. However, the expression, roles and molecular mechanisms of most lncRNAs in glioma are still unknown. In this study, we identified a novel lncRNA LINC00526, which is significantly low expressed in glioma. Low expression of LINC00526 is correlated with aggravation and poor survival in glioma. Functional assays revealed that ectopic expression of LINC00526 inhibits glioma cell proliferation, migration, and invasion. LINC00526 silencing promotes glioma cell proliferation, migration and invasion. Mechanistically, we found that LINC00526 directly interacts with EZH2, represses the binding of EZH2 to AXL promoter, attenuates the transcriptional activating roles of EZH2 on AXL, and therefore represses AXL expression. Via repressing AXL, LINC00526 further represses PI3K/Akt/NF-κB signalling. Intriguingly, we identified that NFKB1 and NFKB2 directly binds LINC00526 promoter and represses LINC00526 transcription. We further found that via activating NF-κB signalling, AXL represses LINC00526 transcription. Therefore, LINC00526/EZH2/AXL/PI3K/Akt/NF-κB form a feedback loop in glioma. Analysis of the TCGA data revealed that the expression of LINC00526 is inversely correlated with that of AXL in glioma tissues. In addition, functional rescue assays revealed that the tumour suppressive roles of LINC00526 are dependent on the negative regulation of AXL. Collectively, our data identified LINC00526 as a tumour suppressor in glioma via forming a double negative feedback loop with AXL. Our data also suggested LINC00526 as a potential prognostic biomarker and therapeutic candidate for glioma.

Siglec-9 is an MHC-independent inhibitory receptor expressed on a subset of natural killer (NK) cells. Siglec-9 restrains NK cytotoxicity by binding to sialoglycans (sialic acid-containing glycans) on target cells. Despite the importance of Siglec-9 interactions in tumor immune evasion, their role as an immune evasion mechanism during HIV infection has not been investigated. Using in vivo phenotypic analyses, we found that Siglec-9+ CD56dim NK cells, during HIV infection, exhibit an activated phenotype with higher expression of activating receptors and markers (NKp30, CD38, CD16, DNAM-1, perforin) and lower expression of the inhibitory receptor NKG2A, compared to Siglec-9- CD56dim NK cells. We also found that levels of Siglec-9+ CD56dim NK cells inversely correlate with viral load during viremic infection and CD4+ T cell-associated HIV DNA during suppressed infection. Using in vitro cytotoxicity assays, we confirmed that Siglec-9+ NK cells exhibit higher cytotoxicity towards HIV-infected cells compared to Siglec-9- NK cells. These data are consistent with the notion that Siglec-9+ NK cells are highly cytotoxic against HIV-infected cells. However, blocking Siglec-9 enhanced NK cells' ability to lyse HIV-infected cells, consistent with the known inhibitory function of the Siglec-9 molecule. Together, these data support a model in which the Siglec-9+ CD56dim NK subpopulation is highly cytotoxic against HIV-infected cells even whilst being restrained by the inhibitory effects of Siglec-9. To harness the cytotoxic capacity of the Siglec-9+ NK subpopulation, which is dampened by Siglec-9, we developed a proof-of-concept approach to selectively disrupt Siglec/sialoglycan interactions between NK and HIV-infected cells. We achieved this goal by conjugating Sialidase to several HIV broadly neutralizing antibodies. These conjugates selectively desialylated HIV-infected cells and enhanced NK cells' capacity to kill them. In summary, we identified a novel, glycan-based interaction that may contribute to HIV-infected cells' ability to evade NK immunosurveillance and developed an approach to break this interaction.

Genetic optimization of Nucleic Acid immunogens is important for potentially improving their immune potency. A COVID-19 DNA vaccine is in phase III clinical trial which is based on a promising highly developable technology platform. Here, we show optimization in mice generating a pGX-9501 DNA vaccine encoding full-length spike protein, which results in induction of potent humoral and cellular immune responses, including neutralizing antibodies, that block hACE2-RBD binding of live CoV2 virus in vitro. Optimization resulted in improved induction of cellular immunity by pGX-9501 as demonstrated by increased IFN-γ expression in both CD8+ and CD4 + T cells and this was associated with more robust antiviral CTL responses compared to unoptimized constructs. Vaccination with pGX-9501 induced subsequent protection against virus challenge in a rigorous hACE2 transgenic mouse model. Overall, pGX-9501 is a promising optimized COVID-19 DNA vaccine candidate inducing humoral and cellular immunity contributing to the vaccine's protective effects.

Milk oligosaccharides (OS) shape microbiome structure and function, but their relative abundances differ between species. Herein, the impact of the human milk oligosaccharides (HMO) (2'-fucosyllactose [2'FL] and lacto-N-neotetraose [LNnT]) and OS isolated from bovine milk (BMOS) on microbiota composition and volatile fatty acid (VFA) concentrations in ascending colon (AC) contents and feces was assessed. Intact male piglets received diets either containing 6.5 g/L BMOS (n = 12), 1.0 g/L 2'FL + 0.5 g/L LNnT (HMO; n = 12), both (HMO + BMOS; n = 10), or neither (CON; n = 10) from postnatal day (PND) 2 to 34. Microbiota were assessed by 16S rRNA gene sequencing and real-time PCR, and VFA were measured by gas chromatography. The microbiota was affected by OS in an intestine region-specific manner. BMOS reduced (p < 0.05) microbial richness in the AC, microbiota composition in the AC and feces, and acetate concentrations in AC, regardless of HMO presence. HMO alone did not affect overall microbial composition, but increased (p < 0.05) the relative proportion of specific taxa, including Blautia, compared to other groups. Bacteroides abundance was increased (p < 0.05) in the AC by BMOS and synergistically by BMOS + HMO in the feces. Distinct effects of HMO and BMOS suggest complementary and sometimes synergistic benefits of supplementing a complex mixture of OS to formula.

Arginase is a complex and unique enzyme that plays diverse roles in health and disease. By metabolizing arginine, it can shape the outcome of innate and adaptive immune responses. The immunomodulatory capabilities of arginase could potentially be applied for local immunosuppression or induction of immune tolerance. With the use of an enhanced DNA delivery approach, we designed and studied a DNA-encoded secretable arginase enzyme as a tool for immune modulation and evaluated its immunomodulatory function in vivo. Strong immunosuppression of cluster of differentiation 4 (CD4) and CD8 T cells, as well as macrophages and dendritic cells, was observed in vitro in the presence of an arginase-rich supernatant. To further evaluate the efficacy of DNA-encoded arginase on in vivo immunosuppression against an antigen, a cancer antigen vaccine model was used in the presence or absence of DNA-encoded arginase. Significant in vivo immunosuppression was observed in the presence of DNA-encoded arginase. The efficacy of this DNA-encoded arginase delivery was examined in a local, imiquimod-induced, psoriasis-like, skin-inflammation model. Pretreatment of animals with the synthetic DNA-encoded arginase led to significant decreases in skin acanthosis, proinflammatory cytokines, and costimulatory molecules in extracted macrophages and dendritic cells. These results draw attention to the potential of direct in vivo-delivered arginase to function as an immunomodulatory agent for treatment of local inflammation or autoimmune diseases.