Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.     

A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB

In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. We show here that beta-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3(-/-) mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. We conclude that in the intestinal epithelium beta-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system.     

Context-dependent responses of terrestrial invertebrates to anthropogenic nitrogen enrichment: A meta-analysis

Anthropogenic increases in nitrogen (N) concentrations in the environment are affecting plant diversity and ecosystems worldwide, but relatively little is known about N impacts on terrestrial invertebrate communities. Here, we performed an exploratory meta-analysis of 4365 observations from 126 publications reporting on the richness (number of taxa) or abundance (number of individuals per taxon) of terrestrial arthropods or nematodes in relation to N addition. We found that the response of invertebrates to N enrichment is highly dependent on both species' traits and local climate. The abundance of arthropods with incomplete metamorphosis, including agricultural pest species, increased in response to N enrichment. In contrast, arthropods exhibiting complete or no metamorphosis, including pollinators and detritivores, showed a declining abundance trend with increasing N enrichment, particularly in warmer climates. These contrasting and context-dependent responses may explain why we detected no overall response of arthropod richness. For nematodes, the abundance response to N enrichment was dependent on mean annual precipitation and varied between feeding guilds. We found a declining trend in abundance with N enrichment in dry areas and an increasing trend in wet areas, with slopes differing between feeding guilds. For example, at mean levels of precipitation, bacterivore abundance showed a positive trend in response to N addition while fungivore abundance declined. We further observed an overall decline in nematode richness with N addition. These N-induced changes in invertebrate communities could have negative consequences for various ecosystem functions and services, including those contributing to human food production.     

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.     

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR^1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10^th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application^4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2^nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats^9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)¹². In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR⁺ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21¹³. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.