Commercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p < 0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

Redox signaling has been established as an essential component of inflammatory responses, and redox active compounds are of interest as potential immunomodulatory agents. Dibenzocyclooctadiene lignans isolated from Schisandra chinensis, a medicinal plant with widespread use in oriental medicine, have been implicated to possess immunomodulatory properties but their effects on the human innate immune system cells have not been described. In this contribution, data are presented on the impact of schisandrin, schisandrin B and schisandrin C on human monocytic cell redox status, as well as their impact on dendritic cell maturation and T cell activation capacity and cytokine production. In THP-1 cells, levels of intracellular reactive oxygen species (ROS) were elevated after 1 h exposure to schisandrin. Schisandrin B and schisandrin C decreased cellular glutathione pools, which is a phenotype previously reported to promote anti-inflammatory functions. Treatment of human primary monocytes with the lignans during their maturation to dendritic cells did not have any effect on the appearance of surface markers HLA-DR and CD86 but schisandrin B and schisandrin C suppressed the secretion of cytokines interleukin (IL)-6, IL-10 and IL-12 by the mature dendritic cells. Dendritic cells maturated in presence of schisandrin C were further cocultured with naïve CD4+ T cells, resulting in reduced IL-12 production. In THP-1 cells, schisandrin B and schisandrin C reduced the IL-6 and IL-12 production triggered by E. coli lipopolysaccharide and IL-12 production induced by an infection with Chlamydia pneumoniae. In conclusion, the studied lignans act as immunomodulatory agents by altering the cytokine secretion, but do not interfere with dendritic cell maturation. And the observed effects may be associated with the ability of the lignans to alter cellular redox status.

The in vivo mechanism of tea polyphenol-mediated prevention of many chronic diseases is still largely unknown. Studies have shown that accumulation of toxic reactive cellular metabolites, such as ammonia and reactive carbonyl species (RCS), is one of the causing factors to the development of many chronic diseases. In this study, we investigated the in vivo interaction between (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenol in tea leaves, and ammonia and RCS. We found that EGCG could be oxidized to EGCG quinone in mice, and then rapidly react with ammonia to generate the aminated EGCG metabolite, 4'-NH2-EGCG. Both EGCG and its aminated metabolite could further scavenge RCS, such as methylglyoxal (MGO), malondialdehyde (MDA), and trans-4-hydroxy-2-nonenal (4-HNE), to produce the RCS conjugates of EGCG and the aminated EGCG. Both the aminated and the RCS conjugated metabolites of EGCG were detected in human after drinking four cups of green tea per day. By comparing the levels of the aminated and the RCS conjugated metabolites in EGCG exposed germ-free (GF) mice and specific-pathogen-free (SPF) mice, we demonstrated that gut microbiota facilitate the formation of the aminated metabolite of EGCG, the RCS conjugates of EGCG, and the RCS conjugates of the aminated EGCG. By comparing the trapping capacities of EGCG and its aminated metabolite under aerobic and anaerobic conditions, we found that oxygen is not essential for the trapping of reactive species by EGCG and 4'-NH2-EGCG suggesting that EGCG and its aminated metabolite could scavenge RCS in the GI track and in the circulation system. Altogether, this study provides in vivo evidences that EGCG has the capacity to scavenge toxic reactive metabolic wastes. This finding opens a new window to understand the underlying mechanisms by which drinking tea could prevent the development of chronic diseases.

In mammals, the cochlear sensory epithelium becomes quiescent early during development. After the first postnatal week, there is no cell replacement or proliferation, and severe damage leads to permanent deafness. Supporting cells' trans-differentiation has been suggested as a way to regenerate cochlear hair cells after damage. However, they are also needed for proper functionality. Cdkn1b (p27Kip1) participates in the cochlear terminal mitosis state achieved during development. Its expression is maintained in adult supporting cells and its postnatal deletion has induced cochlear proliferation in vitro and in vivo. Therefore, its manipulation has been proposed as a feasible way to induce proliferation of supporting cells after birth. Nevertheless, the literature is scarce regarding feasible methods to directly decrease p27Kip1 in the clinical domain. The effects of p27Kip1 knockdown using viral vectors are not completely elucidated and no pharmacological approaches to decrease p27Kip1 in the cochlea have been tested in vivo before. This study explores the ability of p27Kip1 messenger knockdown and pharmacological transcriptional inhibition to induce proliferation of supporting cells in the P0 neonatal rat cochlea in vivo. Respectively, lentiviral vectors transducing shRNA against p27Kip1 were administered into the scala media or Alsterpaullone 2-Cyanoethyl into the round window niche. Cell markers and gene expression were assessed through immunostaining and qRT-PCR. Despite both methods significantly decreasing p27Kip1 expression in vivo, signs of toxicity in the organ of Corti were not found; however, relevant proliferation was not found either. Finally, cochlear damage was added to increase the response in vitro, achieving only a mild to moderate proliferation induction. We conclude that our approaches were not able to stimulate the recall of supporting cell proliferation despite significantly decreased p27Kip1 levels in vivo. Considering the evaluation of the cochlea at a very responsive stage, we propose that the level of isolated modification of p27Kip1 expression in living mammals achievable through these approaches is insufficient to induce proliferation of supporting cells. Future proliferation induction experiments in the cochlea should study other methods and genes.

Large-scale otoscopic and audiometric assessment of populations is difficult due to logistic impracticalities, particularly in low- and middle-income countries (LMIC). We report a novel assessment methodology based on training local field workers, advances in audiometric testing equipment and cloud-based technology.

The European bisons are the largest mammals of Europe that are still in danger of extinction. The species conservation is associated with their continuous reproduction, and bisons are characterized by the well-pronounced seasonality of reproductive processes. However, the exact mechanisms regulating their reproduction still remain unknown. Our previous studies indicated the involvement of some of the growth factors in the regulation of male seasonal reproductive activities in bison, showing expression patterns that seemed to be regulated by the length of the daylight. In the present study, using RT-PCR and Western blot approaches, we verified the expression and possible relationship between the insulin-like growth factor (IGF-1), its receptor (IGF-1R), and klotho in testis and epididymis of the European bison in pre- and post-reproductive periods, i.e., in June and in December. The observed expression of IGF-1 and IGF-1R mRNA in testis and epididymis was higher in June than in December. At the same time, klotho mRNA expression in both testis and epididymis did not differ between the analyzed seasons. However, along with the higher levels of IGF-1R protein observed in June, klotho protein levels for the membrane form and for the secrete form were higher in December than in June. Finally, the messenger and protein expression profiles presented herein indicate the importance of both the IGF-system and klotho in reproductive processes in the European bison, implying their involvement in the regulation of seasonal testicular activity in males of this threatened species.

N-acetyl group metabolites are a general class of endogenous compounds characterized by a conjugated system consisting of an acetyl group and nitrogen moiety. The aim of our exploratory pilot case-control study is to compare the levels of five N-acetyl derivatives (i.e., N-acetyl-glutamine, N-acetyl-ornithine, N6-acetyl-L-lysine, N-acetyl-putrescine, and N-acetyl-galactosamine) in serum samples between individuals with first-episode schizophrenia and healthy controls (HC). A 1:2 age- and sex- matched pilot case-control study was performed, involving 30 cases of first-episode schizophrenia and 60 HC aged between 18 and 40 years old. The serum samples containing these N-acetyl derivatives from (first-episode patients with schizophrenia and HC were measured using liquid chromatography-tandem mass spectrometry (LC-MS). Results indicated that higher levels of N-acetyl-glutamine and lower levels of N6-acetyl-L-lysine may have a significant association with schizophrenia after adjusting for age, sex and BMI. N-acetyl-putrescine was elevated among subjects with first-episode schizophrenia when compared to HC, suggesting it as a predictor for schizophrenia onset. Further exploration of the mechanisms of N-acetyl group metabolites with respect to schizophrenia is warranted and may be useful for identifying novel disease markers and/or drug target molecules in schizophrenia.

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

There are various established scoring systems to assess the outcome of clubfoot treatment after correction with the Ponseti method. We used five measures to compare the results in a cohort of children followed up for between 3.5 to 5 years.

Recent evidence suggests that long non-coding RNAs (lncRNAs) are key regulators in the pathological process of ischemic stroke (IS). Maternally expressed gene 3 (MEG3) was observed to be up-regulated in IS, acting as a competing endogenous RNA for miR-181b to regulate ischemic brain injury. The purpose of this study was to evaluate the association of tagSNPs in MEG3 (i.e., rs7158663 and rs4081134) and miR-181b rs322931 with IS risk.

Trimethylamine N-oxide (TMAO), a small molecule produced by the metaorganismal metabolism of dietary choline, has been implicated in human disease pathogenesis, including known risk factors for Alzheimer's disease (AD), such as metabolic, cardiovascular, and cerebrovascular disease.

The human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. Research reports on the mechanism of HEF1 in gastric cancer (GC) differentiation are limited. In this study, we reveal that HEF1 plays an essential role in regulating of differentiation in human GC. HEF1 was found to be highly expressed in GC tissues. Besides, In GC tissues or cells, cellular level of HEF1 negatively correlated with tumor differentiation. In addition, we showed that upregulation of HEF1 increased Wnt5a expression and the nuclear translocation of β-catenin, thereby resulting in poor differentiation in GC. Notably, GC patients with a higher expression of HEF1 showed significantly poorer disease-free and overall survival. Thus, our findings suggest that HEF1 reduces differentiation through the Wnt5a/β-catenin signaling pathway and that HEF1 is an independent unfavorable prognostic death factor in GC.

Recent evidence provides support for involvement of the microbiota-gut-brain axis in Parkinson's disease (PD) pathogenesis. We propose that a pro-inflammatory intestinal milieu, due to intestinal hyper-permeability and/or microbial dysbiosis, initiates or exacerbates PD pathogenesis. One factor that can cause intestinal hyper-permeability and dysbiosis is chronic stress which has been shown to accelerate neuronal degeneration and motor deficits in Parkinsonism rodent models. We hypothesized that stress-induced intestinal barrier dysfunction and microbial dysbiosis lead to a pro-inflammatory milieu that exacerbates the PD phenotype in the low-dose oral rotenone PD mice model. To test this hypothesis, mice received unpredictable restraint stress (RS) for 12 weeks, and during the last six weeks mice also received a daily administration of low-dose rotenone (10 mg/kg/day) orally. The initial six weeks of RS caused significantly higher urinary cortisol, intestinal hyperpermeability, and decreased abundance of putative "anti-inflammatory" bacteria (Lactobacillus) compared to non-stressed mice. Rotenone alone (i.e., without RS) disrupted the colonic expression of the tight junction protein ZO-1, increased oxidative stress (N-tyrosine), increased myenteric plexus enteric glial cell GFAP expression and increased α-synuclein (α-syn) protein levels in the colon compared to controls. Restraint stress exacerbated these rotenone-induced changes. Specifically, RS potentiated rotenone-induced effects in the colon including: 1) intestinal hyper-permeability, 2) disruption of tight junction proteins (ZO-1, Occludin, Claudin1), 3) oxidative stress (N-tyrosine), 4) inflammation in glial cells (GFAP + enteric glia cells), 5) α-syn, 6) increased relative abundance of fecal Akkermansia (mucin-degrading Gram-negative bacteria), and 7) endotoxemia. In addition, RS promoted a number of rotenone-induced effects in the brain including: 1) reduced number of resting microglia and a higher number of dystrophic/phagocytic microglia as well as (FJ-C+) dying cells in the substantia nigra (SN), 2) increased lipopolysaccharide (LPS) reactivity in the SN, and 3) reduced dopamine (DA) and DA metabolites (DOPAC, HVA) in the striatum compared to control mice. Our findings support a model in which chronic stress-induced, gut-derived, pro-inflammatory milieu exacerbates the PD phenotype via a dysfunctional microbiota-gut-brain axis.

Premigratory neural crest cells arise within the dorsal neural tube and subsequently undergo an epithelial-to-mesenchymal transition (EMT) to leave the neuroepithelium and initiate migration. Draxin is a Wnt modulator that has been shown to control the timing of cranial neural crest EMT. Here we show that this process is accompanied by three stages of remodeling of the basement membrane protein laminin, from regression to expansion and channel formation. Loss of Draxin results in blocking laminin remodeling at the regression stage, whereas ectopic maintenance of Draxin blocks remodeling at the expansion stage. The latter effect is rescued by addition of Snail2, previously shown to be downstream of Draxin. Our results demonstrate an essential function for the Wnt modulator Draxin in regulating basement membrane remodeling during cranial neural crest EMT.

Investigations into hydrogen sulfide (H2S) signaling pathways have demonstrated both the generation and importance of persulfides, which are reactive sulfur species that contain both reduced and oxidized sulfur. These observations have led researchers to suggest that oxidized sulfur species, including sulfane sulfur (S0), are responsible for many of the physiological phenomena initially attributed to H2S. A common method of introducing S0 to biological systems is the administration of organic polysulfides, such as diallyl trisulfide (DATS). However, prior reports have demonstrated that commercially-available DATS often contains a mixture of polysulfides, and furthermore a lack of structure-activity relationships for organic polysulfides has limited our overall understanding of different polysulfides and their function in biological systems. Advancing our interests in the chemical biology of reactive sulfur species including H2S and S0, we report here our investigations into the rates and quantities of H2S release from a series of synthetic, pure benzyl polysulfides, ranging from monosulfide to tetrasulfide. We demonstrate that H2S is only released from the trisulfide and tetrasulfide, and that this release requires thiol-mediated reduction in the presence of cysteine or reduced glutathione. Additionally, we demonstrate the different effects of trisulfides and tetrasulfides on cell proliferation in murine epithelial bEnd.3 cells.

A new series of C-phenyl d-glucitol derivatives was designed and synthesized, and their SGLT1 inhibitory potency and absorbability were evaluated. We also investigated whether kidney drug retention could be avoided by creating molecules with different excretion pathways. To achieve a class of molecules with low absorption and that were excreted in bile, optimized synthesis was performed to bring the ClogP value and the topological polar surface area to within the appropriate ranges. Compounds 34d and 34j were poorly absorbed, but the absorbed compounds were mainly excreted in bile. Thus, smaller amounts of persistent residue in the kidneys were observed. Since 34d exerted a glucose-lowering effect at a dose of 0.3 mg/kg (p.o.) in SD rats, this compound (SGL5213) could be a clinical candidate for the treatment of type 2 diabetes.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that modify extracellular matrix components and play crucial roles in development and numerous diseases. ADAMTS18 is a member of the ADAMTS family, and genome-wide association studies made an initial association of ADAMTS18 with white matter integrity in healthy people of 72-74 years old. However, the potential roles of ADAMTS18 in central nervous system remain unclear. In this study, we showed that Adamts18 mRNA is highly abundant in developing brains, especially in the cerebellum granular cell layer and the hippocampus dentate gyrus (DG) granular cell layer. Adamts18 knockout (KO) mice displayed higher dendritic branching complexity and spine density on hippocampal DG granular cells. Behavioral tests showed that Adamts18 KO mice had reduced levels of depression-like behaviors compared to their wild-type (WT) littermates. The increased neurite formation could be attributed in part to reduced phosphorylation levels of the collapsin response mediator protein-2 (CRMP2) due to activation of the laminin/PI3K/AKT/GSK-3β signaling pathway. Our findings revealed a critical role of ADAMTS18 in neuronal morphogenesis and emotional control in mice.

No abstract available

Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models.

Neointimal hyperplasia is a prominent pathological event during in-stent restenosis. Phenotype switching of vascular smooth muscle cells (VSMCs) from a differentiated/contractile to a dedifferentiated/synthetic phenotype, accompanied by migration and proliferation of VSMCs play an important role in neointimal hyperplasia. However, the molecular mechanisms underlying phenotype switching of VSMCs have yet to be fully understood.