One-pot bio-synthesis: N-acetyl-d-neuraminic acid production by a powerful engineered whole-cell catalyst

Whole cell biocatalysis is an important tool for pharmaceutical intermediates synthesis, although it is hindered by some shortcomings, such as high cost and toxicity of inducer, mass transfer resistance caused by cell membrane and side reactions. Whole-cell catalysis using N-acetyl-d-glucosamine 2-epimerase (EC 5.1.3.8) and N-acetyl-d-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) is a promising approach for the production of Neu5Ac, a potential precursor of many anti-viral drugs. A powerful catalyst was developed by packaging the enzymes in an engineered bacterium and using a safe temperature-induced vector. Since the mass transfer resistance and the side reactions were substantially reduced, a high Neu5Ac amount (191 mM) was achieved. An efficient method was also presented, which allows one-pot synthesis of Neu5Ac with a safe and economic manner. The results highlight the promise of large-scale Neu5Ac synthesis and point at a potential of our approach as a general strategy to improve whole-cell biocatalysis

Unique Changes in Mitochondrial Genomes Associated with Reversions of S-Type Cytoplasmic Male Sterility in Maizemar

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitmate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations

TLR9-Dependent and Independent Pathways Drive Activation of the Immune System by Propionibacterium Acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1–2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders

Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients

Background: Dysregulated expression of Kallikrein-related peptidase 6 (KLK6) is a common feature for many human malignancies and numerous studies evaluated KLK6 as a promising biomarker for early diagnosis or unfavorable prognosis. However, the expression of KLK6 in carcinomas derived from mucosal epithelia, including head and neck squamous cell carcinoma (HNSCC), and its mode of action has not been addressed so far. Methods: Stable clones of human mucosal tumor cell lines were generated with shRNA-mediated silencing or ectopic overexpression to characterize the impact of KLK6 on tumor relevant processes in vitro. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan-Meier and multivariate Cox proportional hazard model analysis. Results: KLK6 expression was detected in head and neck tumor cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. Silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling epithelial-to-mesenchymal transition, nuclear β-catenin accumulation and higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome. Conclusions: In summary, detection of low KLK6 expression in primary tumors represents a promising tool to stratify HNSCC patients with high risk for treatment failure. These patients might benefit from restoration of KLK6 expression or pharmacological targeting of signaling pathways implicated in EMT

Deficient of a Clock Gene, Brain and Muscle Arnt-Like Protein-1 (BMAL1), Induces Dyslipidemia and Ectopic Fat Formation

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome

Aβ Mediated Diminution of MTT Reduction—An Artefact of Single Cell Culture?

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) reduction assay is a frequently used and easily reproducible method to measure beta-amyloid (Aβ) toxicity in different types of single cell culture. To our knowledge, the influence of Aβ on MTT reduction has never been tested in more complex tissue. Initially, we reproduced the disturbed MTT reduction in neuron and astroglia primary cell cultures from rats as well as in the BV2 microglia cell line, utilizing four different Aβ species, namely freshly dissolved Aβ (25-35), fibrillar Aβ (1-40), oligomeric Aβ (1-42) and oligomeric Aβ (1-40). In contrast to the findings in single cell cultures, none of these Aβ species altered MTT reduction in rat organotypic hippocampal slice cultures (OHC). Moreover, application of Aβ to acutely isolated hippocampal slices from adult rats and in vivo intracerebroventricular injection of Aβ also did not influence the MTT reduction in the respective tissue. Failure of Aβ penetration into the tissue cannot explain the differences between single cells and the more complex brain tissue. Thus electrophysiological investigations disclosed an impairment of long-term potentiation (LTP) in the CA1 region of hippocampal slices from rat by application of oligomeric Aβ (1-40), but not by freshly dissolved Aβ (25-35) or fibrillar Aβ (1-40). In conclusion, the experiments revealed a glaring discrepancy between single cell cultures and complex brain tissue regarding the effect of different Aβ species on MTT reduction. Particularly, the differential effect of oligomeric versus other Aβ forms on LTP was not reflected in the MTT reduction assay. This may indicate that the Aβ oligomer effect on synaptic function reflected by LTP impairment precedes changes in formazane formation rate or that cells embedded in a more natural environment in the tissue are less susceptible to damage by Aβ, raising cautions against the consideration of single cell MTT reduction activity as a reliable assay in Alzheimer's drug discovery studies

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract

Childhood sarcoidosis: A rare but fascinating disorder

Childhood sarcoidosis is a rare multisystemic granulomatous disorder of unknown etiology. In the pediatric series reported from the southeastern United States, sarcoidosis had a higher incidence among African Americans. Most reported childhood cases have occurred in patients aged 13–15 years. Macrophages bearing an increased expression of major histocompatibility class (MHC) II molecules most likely initiate the inflammatory response of sarcoidosis by presenting an unidentified antigen to CD4+ Th (helper-inducer) lymphocytes. A persistent, poorly degradable antigen driven cell-mediated immune response leads to a cytokine cascade, to granuloma formation, and eventually to fibrosis. Frequently observed immunologic features include depression of cutaneous delayed-type hypersensitivity and a heightened helper T cell type 1 (Th1) immune response at sites of disease. Circulating immune complexes, along with signs of B cell hyperactivity, may also be found. The clinical presentation can vary greatly depending upon the organs involved and age of the patient. Two distinct forms of sarcoidosis exist in children. Older children usually present with a multisystem disease similar to the adult manifestations, with frequent hilar lymphadenopathy and pulmonary infiltrations. Early-onset sarcoidosis is a unique form of the disease characterized by the triad of rash, uveitis, and arthritis in children presenting before four years of age. The diagnosis of sarcoidosis is confirmed by demonstrating a typical noncaseating granuloma on a biopsy specimen. Other granulmatous diseases should be reasonably excluded. The current therapy of choice for sarcoidosis in children with multisystem involvement is oral corticosteroids. Methotrexate given orally in low doses has been effective, safe and steroid sparing in some patients. Alternative immunosuppressive agents, such as azathioprine, cyclophosphamide, chlorambucil, and cyclosporine, have been tried in adult cases of sarcoidosis with questionable efficacy. The high toxicity profile of these agents, including an increased risk of lymphoproliferative disorders and carcinomas, has limited their use to patients with severe disease refractory to other agents. Successful steroid sparing treatment with mycophenolate mofetil was described in an adolescent with renal-limited sarcoidosis complicated by renal failure. Novel treatment strategies for sarcoidosis have been developed including the use of TNF-alpha inhibitors, such as infliximab. The long-term course and prognosis is not well established in childhood sarcoidosis, but it appears to be poorer in early-onset disease

Experimental traumatic brain injury

Traumatic brain injury, a leading cause of death and disability, is a result of an outside force causing mechanical disruption of brain tissue and delayed pathogenic events which collectively exacerbate the injury. These pathogenic injury processes are poorly understood and accordingly no effective neuroprotective treatment is available so far. Experimental models are essential for further clarification of the highly complex pathology of traumatic brain injury towards the development of novel treatments. Among the rodent models of traumatic brain injury the most commonly used are the weight-drop, the fluid percussion, and the cortical contusion injury models. As the entire spectrum of events that might occur in traumatic brain injury cannot be covered by one single rodent model, the design and choice of a specific model represents a major challenge for neuroscientists. This review summarizes and evaluates the strengths and weaknesses of the currently available rodent models for traumatic brain injury

Flavonoids in prevention of diseases with respect to modulation of Ca-pump function

Flavonoids, natural phenolic compounds, are known as agents with strong antioxidant properties. In many diseases associated with oxidative/nitrosative stress and aging they provide multiple biological health benefits. Ca2+-ATPases belong to the main calcium regulating proteins involved in the balance of calcium homeostasis, which is impaired in oxidative/nitrosative stress and related diseases or aging. The mechanisms of Ca2+-ATPases dysfunction are discussed, focusing on cystein oxidation and tyrosine nitration. Flavonoids act not only as antioxidants but are also able to bind directly to Ca2+-ATPases, thus changing their conformation, which results in modulation of enzyme activity