Process analytical utility of Raman spectroscopy for cell therapy manufacturing

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Stability, Entrapment and Variant Formation of Salmonella Genomic Island 1

<div><h3>Background</h3><p>The <em>Salmonella</em> genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in <em>S</em>. Typhimurium.</p> <h3>Methodology/Principal Findings</h3><p>Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in <em>Salmonella</em> populations of 17 <em>Salmonella enterica</em> serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the <em>E. coli</em> chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.</p> <h3>Conclusions/Significance</h3><p>Despite that excision of SGI1 from the chromosome was proven in SGI1⁺<em>Salmonella</em> populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.</p> </div

Transformation and analysis of tobacco plant var Petit havana with T-urf13 gene under anther-specific TA29 promoter

T-urf13, a well-documented cms-associated gene from maize, has been shown to render methomyl sensitivity to heterologous systems like rice, yeast and bacteria when expressed constitutively. Since these transgenic plants were fertile, it was hypothesized that T-urf13 gene if expressed in anthers may result in male sterility that could be used for hybrid seed production. Hence, this work was aimed at analysing whether T-urf13 gene when expressed in anthers can result in male sterile plants or requires methomyl treatment to cause male sterility (controllable). This is the first report of transformation of tobacco with T-urf13 gene under anther-specific promoter (TA29) with or without mitochondrial targeting sequence. Most of the transgenic plants obtained were fertile; this was surprising as many male sterile plants were expected as T-urf13 gene is a cms associated gene. Our results suggest that it may not be possible to obtain male sterility by expressing URF13 in the anther by itself or by methomyl application

Relationship between propagule pressure and colonization pressure in invasion ecology: a test with ships' ballast

Increasing empirical evidence indicates the number of released individuals (i.e. propagule pressure) and number of released species (i.e. colonization pressure) are key determinants of the number of species that successfully invade new habitats. In view of these relationships, and the possibility that ships transport whole communities of organisms, we collected 333 ballast water and sediment samples to investigate the relationship between propagule and colonization pressure for a variety of diverse taxonomic groups (diatoms, dinoflagellates and invertebrates). We also reviewed the scientific literature to compare the number of species transported by ships to those reported in nature. Here, we show that even though ships transport nearly entire local communities, a strong relationship between propagule and colonization pressure exists only for dinoflagellates. Our study provides evidence that colonization pressure of invertebrates and diatoms may fluctuate widely irrespective of propagule pressure. We suggest that the lack of correspondence is explained by reduced uptake of invertebrates into the transport vector and the sensitivity of invertebrates and diatoms to selective pressures during transportation. Selection during transportation is initially evident through decreases in propagule pressure, followed by decreased colonization pressure in the most sensitive taxa

Dynamics of the Leaf-Litter Arthropod Fauna Following Fire in a Neotropical Woodland Savanna

Fire is an important agent of disturbance in tropical savannas, but relatively few studies have analyzed how soil-and-litter dwelling arthropods respond to fire disturbance despite the critical role these organisms play in nutrient cycling and other biogeochemical processes. Following the incursion of a fire into a woodland savanna ecological reserve in Central Brazil, we monitored the dynamics of litter-arthropod populations for nearly two years in one burned and one unburned area of the reserve. We also performed a reciprocal transplant experiment to determine the effects of fire and litter type on the dynamics of litter colonization by arthropods. Overall arthropod abundance, the abundance of individual taxa, the richness of taxonomic groups, and the species richness of individual taxa (Formiciade) were lower in the burned site. However, both the ordinal-level composition of the litter arthropod fauna and the species-level composition of the litter ant fauna were not dramatically different in the burned and unburned sites. There is evidence that seasonality of rainfall interacts with fire, as differences in arthropod abundance and diversity were more pronounced in the dry than in the wet season. For many taxa the differences in abundance between burned and unburned sites were maintained even when controlling for litter availability and quality. In contrast, differences in abundance for Collembola, Formicidae, and Thysanoptera were only detected in the unmanipulated samples, which had a lower amount of litter in the burned than in the unburned site throughout most of our study period. Together these results suggest that arthropod density declines in fire-disturbed areas as a result of direct mortality, diminished resources (i.e., reduced litter cover) and less favorable microclimate (i.e., increased litter desiccation due to reduction in tree cover). Although these effects were transitory, there is evidence that the increasingly prevalent fire return interval of only 1–2 years may jeopardize the long-term conservation of litter arthropod communities

Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium bifidum Z9. L. acidophilus NCFM strongly induced expression of interferon (IFN)-β, other virus defence genes, and cytokine and chemokine genes related to the innate and the adaptive immune response. By contrast, B. bifidum Z9 up-regulated genes encoding cytokines and chemokines related to the innate immune response. Moreover, B. bifidum Z9 inhibited the expression of the Th1-promoting genes induced by L. acidophilus NCFM and had an additive effect on genes of the innate immune response and Th2 skewing genes. The gene encoding Jun dimerization protein 2 (JDP2), a transcription factor regulating the activation of JNK, was one of the few genes only induced by B. bifidum Z9. Neutralization of IFN-β abrogated L. acidophilus NCFM-induced expression of Th1-skewing genes, and blocking of the JNK pathway completely inhibited the expression of IFN-β. Our results indicate that B. bifidum Z9 actively inhibits the expression of genes related to the adaptive immune system in murine dendritic cells and that JPD2 via blocking of IFN-β plays a central role in this regulatory mechanism

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies

Change in Sediment Features and the Macroinvertebrate Community Within an Estuarine Ecosystem Two Years Post‐restoration

Our objective was to assess the response of an estuarine ecosystem to restoration efforts, two years post‐restoration. Sediment attributes of particle size distribution (PSD), %LOI, water content and amounts of fine wood debris (FWD), and the macroinvertebrate community were compared among three sites, two reference and the recently restored site. The restored region had been previously used as a log sorting facility. As indicated by PSD, the restored site showed signs of recovery. However, the macroinvertebrate community had still not responded to restoration efforts. Sediments of reference sites were comprised of fine sand, and the macroinvertebrate community was dominated by&nbsp;Macoma&nbsp;spp. By contrast, at the restored site, sediments were mainly comprised of silt followed by fine sand,&nbsp;Macoma&nbsp;spp. was absent, and the main macroinvertebrate was&nbsp;Glycera americana, a polychaeta characteristic of disturbed regions. The restored site still contained significance amounts of FWD as compared to the two reference sites attributed to its previous use. Although still early in its recovery stage, active restoration did have a positive effect and will have likely kick started the region toward recovery and further follow‐up in five years is recommended

Activated Human CD4+CD45RO+ Memory T-Cells Indirectly Inhibit NLRP3 Inflammasome Activation through Downregulation of P2X7R Signalling

Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1β. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS