A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C₄ carboxylates into two molecules of acetyl-CoA without loss of CO₂. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.     

Antimalarial activity of compounds comprising a primary benzene sulfonamide fragment

Despite the urgent need for effective antimalarial drugs with novel modes of action no new chemical class of antimalarial drug has been approved for use since 1996. To address this, we have used a rational approach to investigate compounds comprising the primary benzene sulfonamide fragment as a potential new antimalarial chemotype. We report the in vitro activity against Plasmodium falciparum drug sensitive (3D7) and resistant (Dd2) parasites for a panel of fourteen primary benzene sulfonamide compounds. Our findings provide a platform to support the further evaluation of primary benzene sulfonamides as a new antimalarial chemotype, including the identification of the target of these compounds in the parasite.     

THE INFLUENCE OF MAGNETIC FIELDS ON THE PRIMARY HEALING OF INCISIONAL WOUNDS IN RATS

Our studies were conducted to examine the influence of extremely low frequency magnetic fields (ELF-MF) on skin wound healing in male adults rats. We used 40 Hz and 10 mT sinusoidal fields. We evaluated the rate of wound healing by determining the tissue hydroxyproline concentration and scar imaging in electron microscope. The systemic body response to ELF-MF was detected by analysis of blood morphological and biochemical parameters, such as: RBC, WBC, hemoglobin, hematocrit, reticulocytes, electrolytes, urea, and total protein concentration.

ELF-MF induced the increase of hydroxyproline level in scar tissue and intensified the maternity of collagen seen in the electron microscope. The increase of reticulocyte number in blood confirmed that the healing process in experimental animals was supported by the activation of the oxygen supply and utilization processes, as a result of erythropoietic intensification, without simultaneously upsetting cellular energetic processes. We did not obtain changes in biochemical parameters in blood, such as: electrolytes, urea, and total protein concentration, so we concluded that ELF-MF evoked no negative systemic response.     

Constitutive NF-kappaB activation confers interleukin 6 (IL6) independence and resistance to dexamethasone and Janus kinase inhibitor INCB018424 in murine plasmacytoma cells

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.     

TAF4b promotes mouse primordial follicle assembly and oocyte survival

Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development.     

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.     

Hyperactivity,concentration difficulties and impulsiveness improve during seven weeks’ treatment with valerian root and lemon balm extracts in primary school children

BackgroundValerian root and lemon balm extracts have previously shown efficacy and excellent tolerability in children < 12 years suffering from restlessness and insomnia. We now examined whether treatment with a fixed combination of both may also improve concentration, hyperactivity and impulsiveness.Methods169 primary school children suffering from hyperactivity and concentration difficulties but not meeting ADHD criteria were treated in an observational study by 27 office based pediatricians with a recommended daily dose of 640 mg valerian root extract WS® 1014 and 320 mg lemon balm extract WS® 1303 (Sandrin®), and evaluated by pediatricians and parents using standardized questionnaires at baseline, weeks 2 and 7.ResultsThe fraction of children having strong/very strong symptoms of poor ability to focus decreased from 75% to 14%, hyperactivity from 61% to 13%, and impulsiveness from 59% to 22%. Parent rated social behavior, sleep and symptom burden showed highly significant improvements. Only in two children mild transient adverse drug reactions were observed.ConclusionIn primary school children with restlessness, concentration difficulties and impulsiveness treatment with WS® 1014 and WS® 1303 (Sandrin®) provides a viable option in addition to counseling and education.     

Anatomical variations in primary teeth microelements with known differences in lead content by micro-Synchrotron Radiation X-Ray Fluorescence (μ-SRXRF) – A preliminary study

Shed teeth have been proposed as trace element biomarkers. This study determined variations in the spatial distribution of Ca, K, Zn, Pb, Mn, Cu, and Sr in four anatomical locations: superficial enamel (SE, 0–10 μm), subsuperficial enamel (SSE, 10–30 μm), primary dentin (PD), and secondary dentin (SD). Five primary incisors were analyzed by micro Synchrotron Radiation X-Ray Fluorescence (μ-SRXRF). Two teeth had low concentrations of lead in the SE (<250 μg/g), while three contained very high lead concentrations in the SE (>2000 μg/g). Teeth were sliced, and five spot measurements (20 μm beam diameter) were accomplished in each location. The data are shown as absolute values and as the ratio between the different elements and Ca. The distribution of K was close to that of Ca. Zn was the third most abundant element, with the highest levels being found in the SE and SD and low levels detected in the PD. Increasing Sr levels were found progressing from the enamel to the dentin, with the highest levels being found in the SD, a distribution that was unique. Pb, Mn, and Cu exhibited a similar trend, with higher signals for these elements detected in the SE. This study provides preliminary data on the heterogeneous distribution of different elements in the tooth, highlighting the importance of the first 10 μm of the SE for determination of some elements, such as Zn, Pb, Mn, and Cu.