2013 REU Poster: Purification and Characterization of a Ferredoxin from Mycobacterium tuberculosis

Poster presentation at REU Summer's End Research Symposium, 2013, by REU participant Jonas A, de Oliveira, MassBay Community College - Sean Elliott group, Evan Judd lab mentorM. tuberculosis possesses a sulfite reductase (MtsirA) that is over-expressed when the bacteria is in its dormant stage of infection. MtSirA catalyzes the six-electron reduction of sulfite to sulfide. Previous kinetic studies of MtsirA have used methyl viologen (MV), a chemical reductant, as an electron donor. The goal of this work is to purify and characterize a ferredoxin from M. tuberculosis (MtFd) and determine if MtFd can act as an electron donor to mtSirA, with the ultimate goal of using it as a more physiologically relevant electron donor in kinetic studies of mtSirA. We have found that that MtFd purifies without a cluster and must be chemically reconstituted. MtFd likely contains a [4Fe-4S] cluster, and may be able to donate electrons to mtSirA.NSF-RE

A determination of the Spectra of Galactic components observed by WMAP

WMAP data when combined with ancillary data on free-free, synchrotron and dust allow an improved understanding of the spectrum of emission from each of these components. Here we examine the sky variation at intermediate latitudes using a cross-correlation technique. In particular, we compare the observed emission in 15 selected sky regions to three ``standard'' templates. The free-free emission of the diffuse ionised gas is fitted by a well-known spectrum at K and Ka band, but the derived emissivity corresponds to a mean electron temperature of ~4000-5000K. This is inconsistent with estimates from galactic HII regions. The origin of the discrepancy is unclear. The anomalous emission associated with dust is clearly detected in most of the 15 fields studied; it correlates well with the Finkbeiner et al. model 8 predictions (FDS8) at 94 GHz, with an effective spectral index between 20 and 60GHz of -2.85. Furthermore, the emissivity varies by a factor of ~2 from cloud to cloud. A modestly improved fit to the anomalous dust at K-band is provided by modulating the template by an estimate of the dust colour temperature, specifically FDS8*T^n. We find a preferred value n~1.6. The synchrotron emission steepens between GHz frequencies and the WMAP bands. There are indications of spectral index variations across the sky but the current data are not precise enough to accurately quantify this from region to region. Our analysis of the WMAP data indicates strongly that the dust-correlated emission at the low WMAP frequencies has a spectrum which is compatible with spinning dust; we find no evidence for a synchrotron component correlated with dust (abridged).Comment: 18 pages, 6 figures, revised version uses cross-correlation method rather than T-T method. Paper re-organised and sent back to refere

IR-correlated 31 GHz radio emission from Orion East

Lynds dark cloud LDN1622 represents one of the best examples of anomalous dust emission, possibly originating from small spinning dust grains. We present Cosmic Background Imager (CBI) 31 GHz data of LDN1621, a diffuse dark cloud to the north of LDN1622 in a region known as Orion East. A broken ring with diameter g\approx 20 arcmin of diffuse emission is detected at 31 GHz, at \approx 20-30 mJy beam$^{-1}$ with an angular resolution of \approx 5 arcmin. The ring-like structure is highly correlated with Far Infra-Red emission at $12-100 \mu$m with correlation coefficients of r \approx 0.7-0.8, significant at $\sim10\sigma$. Multi-frequency data are used to place constraints on other components of emission that could be contributing to the 31 GHz flux. An analysis of the GB6 survey maps at 4.85 GHz yields a $3\sigma$ upper limit on free-free emission of 7.2 mJy beam$^{-1}$ (\la 30 per cent of the observed flux) at the CBI resolution. The bulk of the 31 GHz flux therefore appears to be mostly due to dust radiation. Aperture photometry, at an angular resolution of 13 arcmin and with an aperture of diameter 30 arcmin, allowed the use of IRAS maps and the {\it WMAP} 5-year W-band map at 93.5 GHz. A single modified blackbody model was fitted to the data to estimate the contribution from thermal dust, which amounts to \sim$10 per cent at 31 GHz. In this model, an excess of 1.52\pm 0.66 Jy (2.3\sigma) is seen at 31 GHz. Future high frequency$\sim$100-1000 GHz data, such as those from the {\it Planck} satellite, are required to accurately determine the thermal dust contribution at 31 GHz. Correlations with the IRAS$100 \mu$m gave a coupling coefficient of$18.1\pm4.4 \mu$K (MJy/sr)$^{-1}$, consistent with the values found for LDN1622.Comment: 8 pages, 3 figures, 3 tables, submitted to MNRA

Neurodifferentiation and neuroprotection potential of mesenchymal stromal cell-derived secretome produced in different dynamic systems

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-β), osteopontin, nerve growth factor beta (NGFβ), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.This work has been funded by la Caixa Foundation and Portuguese Foundation for Science and Technology (FCT) under the agreement LCF/PR/HP20/52300001; ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020. CRM was supported by a Ph.D. scholarship from FCT and the company Stemmatters, Biotecnologia e Medicina Regenerativa SA (PD/BDE/127833/2016). Funding received by iBB-Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2020) and through the project PTDC/EQU-EQU/31651/2017 is acknowledged. MAF was supported by a Ph.D. scholarship from FCT (SFRH/PD/BD/128328/2017). RC was supported by the EXOpro project (PTDC/EQU-QUE/31651/2017). JPS was supported by a Ph.D. scholarship from FCT and the company Bn’ML—Behavioral & Molecular Lab (PD/BDE/127834/2016). DS was supported by a Ph.D. scholarship from FCT and the company Stemmatters, Biotecnologia e Medicina Regenerativa S.A. (PD/BDE/135567/2018) JC was supported by a Ph.D. scholarship from FCT (SFRH/BD/5813/2020)

Angular Spectra of Polarized Galactic Foregrounds

It is believed that magnetic field lines are twisted and bend by turbulent motions in the Galaxy. Therefore, both Galactic synchrotron emission and thermal emission from dust reflects statistics of Galactic turbulence. Our simple model of Galactic turbulence, motivated by results of our simulations, predicts that Galactic disk and halo exhibit different angular power spectra. We show that observed angular spectra of synchrotron emission are compatible with our model. We also show that our model is compatible with the angular spectra of star-light polarization for the Galactic disk. Finally, we discuss how one can estimate polarized microwave emission from dust in the Galactic halo using star-light polarimetry.Comment: 11 pages, 1 figure; To be published in the proceedings of "The Cosmic Microwave Background and its Polarization", New Astronomy Reviews, (eds. S. Hanany and K.A. Olive

In vitro leishmanicidal, antibacterial and antitumour potential of anhydrocochlioquinone A obtained from the fungus Cochliobolus sp

The bioassay-guided fractionation of the ethyl acetate extract of the fungus Cochliobolus sp. highlighted leishmanicidal activity and allowed for anhydrocochlioquinone A (ANDC-A) isolation. MS, 1D and 2D NMR spectra of this compound were in agreement with those published in the literature. ANDC-A exhibited leishmanicidal activity with EC50value of 22.4 \uc2\ub5g/mL (44 \uce\ubcM) and, when submitted to the microdilution assay against Gram-positive and Gram-negative bacteria, showed a minimal inhibitory concentration against Staphylococcus aureus ATCC 25295 of 128 \uce\ubcg/mL (248.7 \uce\ubcM). It was also active against five human cancer cell lines, showing IC50values from 5.4 to 20.3 \uce\ubcM. ANDC-A demonstrated a differential selectivity for HL-60 (SI 5.5) and THP-1 (SI 4.3) cell lines in comparison with Vero cells and was more selective than cisplatin and doxorubicin against MCF-7 cell line in comparison with human peripheral blood mononuclear cells. ANDC-A was able to eradicate clonogenic tumour cells at concentrations of 20 and 50 \uce\ubcM and induced apoptosis in all tumour cell lines at 20 \uce\ubcM. These results suggest that ANDC-A might be used as a biochemical tool in the study of tumour cells biochemistry as well as an anticancer agent with durable effects on tumours