Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

The Effect of Longterm Exposure to Mercury on the Bacterial Community in Marine Sediment

The aim of this study was to investigate the effect of mercury contamination on bacterial community structure and function. Bacterial communities from two sites, a mercury-contaminated site inside the harbor of Copenhagen, Denmark (CH) and a unpolluted control site, K?ge Buge (KB), were compared with respect to diversity indices, of antibiotic- and heavy metal-resistance patterns, abundance and self transmissibility of plasmids in resistant isolates (endogenous isolation). Furthermore, the potential for gene transfer between indigenous bacteria was assessed by the exogenous plasmid isolation approach.  It was found that resistance to all the tested compounds was higher in the mercury-polluted sediment than the control sediment. The abundance of plasmids was higher at the polluted site, where 62% of the isolates contained plasmids, whereas only 29% of the isolates from the control sediment contained plasmids. Furthermore, the frequencies of large plasmids and plasmids per isolates were found to be higher in the contaminated sediment. Exogenous plasmid isolations revealed high occurrence of Hg and tetracycline resistance, self-transmissible plasmids in CH sediment (1.8 × 10⁻⁵ transconjugants per recipients) relative to KB sediment (3.0 × 10⁻⁸ T/R). Shannon-Weaver diversity indices showed no difference in the diversity of the isolates from the two sites, and Hg-resistant isolates from CH were found to be as diverse as the CH isolates in total. This may be owing to high level of self-transmissible Hg resistance plasmids found in CH. Received: 25 September 1997 / Accepted: 3 November 1997     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

Temperature responses of photosynthetic capacity parameters were not affected by foliar nitrogen content in mature Pinus sylvestris

A key weakness in current Earth System Models is the representation of thermal acclimation of photosynthesis in response to changes in growth temperatures. Previous studies in boreal and temperate ecosystems have shown leaf‐scale photosynthetic capacity parameters, the maximum rates of carboxylation (V_cmax) and electron transport (J_max), to be positively correlated with foliar nitrogen (N) content at a given reference temperature. It is also known that V_cmax and J_max exhibit temperature optima that are affected by various environmental factors and, further, that N partitioning among the foliar photosynthetic pools is affected by N availability. However, despite the strong recent anthropogenic influence on atmospheric temperatures and N deposition to forests, little is known about the role of foliar N contents in controlling the photosynthetic temperature responses. In this study, we investigated the temperature dependencies of V_cmax and J_max in 1‐year‐old needles of mature boreal Pinus sylvestris (Scots pine) trees growing under low and high N availabilities in northern Sweden. We found that needle N status did not significantly affect the temperature responses of V_cmax or J_max when the responses were fitted to a peaked function. If such N insensitivity is a common tree trait it will simplify the interpretation of the results from gradient and multi‐species studies, which commonly use sites with differing N availabilities, on temperature acclimation of photosynthetic capacity. Moreover, it will simplify modeling efforts aimed at understanding future carbon uptake by precluding the need to adjust the shape of the temperature response curves to variation in N availability.     

Bacillus thuringiensis in fecal samples from greenhouse workers after exposure to B. thuringiensis-based pesticides

In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10(2) to 10(3) CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K(+) influx is neither inhibited nor stimulated by Na(+). Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na(+) transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance.     

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Generalized parametric cure models for relative survival

Cure models are used in time-to-event analysis when not all individuals are expected to experience the event of interest, or when the survival of the considered individuals reaches the same level as the general population. These scenarios correspond to a plateau in the survival and relative survival function, respectively. The main parameters of interest in cure models are the proportion of individuals who are cured, termed the cure proportion, and the survival function of the uncured individuals. Although numerous cure models have been proposed in the statistical literature, there is no consensus on how to formulate these. We introduce a general parametric formulation of mixture cure models and a new class of cure models, termed latent cure models, together with a general estimation framework and software, which enable fitting of a wide range of different models. Through simulations, we assess the statistical properties of the models with respect to the cure proportion and the survival of the uncured individuals. Finally, we illustrate the models using survival data on colon cancer, which typically display a plateau in the relative survival. As demonstrated in the simulations, mixture cure models which are not guaranteed to be constant after a finite time point, tend to produce accurate estimates of the cure proportion and the survival of the uncured. However, these models are very unstable in certain cases due to identifiability issues, whereas LC models generally provide stable results at the price of more biased estimates.