Biofinder detects biological remains in Green River fish fossils from Eocene epoch at video speed.

The "Search for life", which may be extinct or extant on other planetary bodies is one of the major goals of NASA planetary exploration missions. Finding such evidence of biological residue in a vast planetary landscape is an enormous challenge. We have developed a highly sensitive instrument, the "Compact Color Biofinder", which can locate minute amounts of biological material in a large area at video speed from a standoff distance. Here we demonstrate the efficacy of the Biofinder to detect fossils that still possess strong bio-fluorescence signals from a collection of samples. Fluorescence images taken by the Biofinder instrument show that all Knightia spp. fish fossils analysed from the Green River formation (Eocene, 56.0-33.9 Mya) still contain considerable amounts of biological residues. The biofluorescence images support the fact that organic matter has been well preserved in the Green River formation, and thus, not diagenetically replaced (replaced by minerals) over such a significant timescale. We further corroborated results from the Biofinder fluorescence imagery through Raman and attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopies, scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM-EDS), and fluorescence lifetime imaging microscopy (FLIM). Our findings confirm once more that biological residues can survive millions of years, and that using biofluorescence imaging effectively detects these trace residues in real time. We anticipate that fluorescence imaging will be critical in future NASA missions to detect organics and the existence of life on other planetary bodies.

Compact Color Biofinder (CoCoBi): Fast, Standoff, Sensitive Detection of Biomolecules and Polyaromatic Hydrocarbons for the Detection of Life.

We have developed a compact instrument called the "COmpact COlor BIofinder", or CoCoBi, for the standoff detection of biological materials and organics with polyaromatic hydrocarbons (PAHs) using a nondestructive approach in a wide area. The CoCoBi system uses a compact solid state, conductively cooled neodymium-doped yttrium aluminum garnet (Nd:YAG) nanosecond pulsed laser capable of simultaneously providing two excitation wavelengths, 355 and 532 nm, and a compact, sensitive-gated color complementary metal-oxide-semiconductor camera detector. The system is compact, portable, and determines the location of biological materials and organics with PAHs in an area 1590 cm2 wide, from a target distance of 3 m through live video using fast fluorescence signals. The CoCoBi system is highly sensitive and capable of detecting a PAH concentration below 1 part per billion from a distance of 1 m. The color images provide the simultaneous detection of various objects in the target area using shades of color and morphological features. We demonstrate that this unique feature successfully detected the biological remains present in a 150-million-year-old fossil buried in a fluorescent clay matrix. The CoCoBi was also successfully field-tested in Hawaiian ocean water during daylight hours for the detection of natural biological materials present in the ocean. The wide-area and video-speed imaging capabilities of CoCoBi for biodetection may be highly useful in future NASA rover-lander life detection missions.

An inexpensive robotic gantry to screen and control soil moisture for plant experiments.

Controlling water content in soil is a recurrent and labor intensive operation on almost any experiment about plant physiology. Here we describe a robotic gantry to measure and control soil moisture in pots that is modular, inexpensive, easy to build, accurate, precise, and reliable. Machines can be stacked into industrial shelves, coupled with other control systems to conduct multifactorial experiments, and adjusted to accommodate numerous pots of any size allowing for experiments with limitless specimen capacity in terms of height and specimen count. The system can be assembled in up to seven hours using off the shelf components and simple tools at a total cost of $1,276, in 2019 prices. A screening cycle can be performed as fast as every six minutes reducing variations in water content due to evaporation and thus creating precise control of soil moisture. As a validation of the long-term cyclic reliability of the system, the machine was run non-stop for 4480 loops; the equivalent to running an experiment for six months controlling water content every hour. By facilitating high throughput monitoring of soil moisture in pots, reliably and at low cost, this machine can facilitate the development of large-scale experiments on plant physiology.

Comprehensive temperature controller with internet connectivity for plant growth experiments.

The experimental control of temperature is key to many scientific and applied endeavors, particularly for studying the effects of greenhouse-gas driven warming on plant performance. Unfortunately, numerous nuisances in the control of temperature for plants renders most commercially available controllers unsuitable or too expensive. Here we describe a simple to use but comprehensive temperature controller for plant growth experiments in enclosed spaces like nurseries or chambers. The device uses Pulse Width Modulation to control independent cooling and heating elements over a broad range of amperages, which minimizes or eliminates temperature overshoot and ensures precise and accurate temperature control (i.e., sensor accuracy = 0.1 °C; controller accuracy = 0.3 °C.). The device incorporates an internal clock for controlling temperature (and growth lights) concurrent with diurnal cycles, and it has an integrated Wi-Fi chip to transfer sensor data to a web-page, where data are displayed in real time. The device uses off-the-shelf parts and can be built for around $USD63. The controller can be integrated with other reported controllers (e.g., soil moisture and CO2) to produce an affordable multi-system controller necessary for complex factorial experiments, which hopefully can help to accelerate our understanding about the impacts of climatic variables on plant performance.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Effects of triazine herbicides on organophosphate insecticide toxicity in Hyalella azteca.

The frequent use of pesticides in agricultural and commercial settings has led some researchers to devote their attention to studying the effects of mixtures of these compounds as they co-occur in the environment. Recent studies have demonstrated the potentiating effects of triazine herbicides, such as atrazine and its analogs, to the toxicity of a variety of organophosphate (OP) insecticides. One such OP insecticide, chlorpyrifos, has been the topic of much concern because of its prevalence in the environment. This study focused on examining the effects of 10 select triazine herbicides at concentrations of 1 mumole/L (approximately 200 mug/L) to chlorpyrifos with Hyalella azteca. The compounds selected include atrazine, three of its degradation products, and six other herbicide active ingredients. Toxicity tests were performed using a two-way analysis of variance matrix design with effect levels determined by way of probit analysis. Atrazine was found to have the greatest acutely lethal effect to H. azteca, followed by its closest degradation product, deethylatrazine. Two of the six atrazine analogs, simazine and cyanazine, also showed significant effects to the insecticide's toxicity. Synergistic ratios (SRs) were calculated to compare the effect magnitudes for each of the herbicides. The highest ratio obtained was with atrazine (SR = 1.42). A majority of the past studies involving mixtures of triazines and OPs have examined the potentiation effects of active-use triazine herbicides on Chironomus species. However, compared with the acute effects previously obtained for Chironomus species, H. azteca show a higher tolerance to the presence of the triazine herbicides, even at levels often considered as being at the high end of environmentally relevant concentrations. When coupled with past studies from our laboratory, this research helps to provide a better understanding of the toxic effects of herbicide-insecticide interactions.

Joint toxicity of triazine herbicides and organophosphate insecticides to the midge Chironomus tentans.

A series of recent studies demonstrated that the triazine herbicide atrazine, although not itself acutely toxic, potentiated the toxicity of certain organophosphate insecticides (OPs) to the midge Chironomus tentans. In the current study, a series of triazine herbicides and triazine herbicide degradation products were tested to determine if other triazines potentiate OP toxicity to midges. Chlorpyrifos and diazinon were the OPs tested. Toxicity tests were conducted using a factorial design and analysis of variance to statistically determine if each triazine had an effect on expected toxicity. Log-probit procedures were also used to evaluate the magnitude of change in median effective concentration (EC50) values during coexposure with each triazine. All of the triazine herbicides tested (atrazine, simazine, cyanazine, and hexazinone) were capable of potentiating the toxicity of the OPs, whereas the degradation products (s-triazine, deethylatrazine, and deisopropylatrazine) had less effect. In most cases, a triazine concentration of 100 microg/L was necessary to significantly increase OP toxicity, and higher concentrations of triazine caused a greater degree of potentiation. Changes in EC50 values ranged from no change to a 2.5-fold increase in toxicity. Generally, EC50 values changed by less than a factor of 2, indicating that the effect may be of limited concern in regard to future risk assessments of OPs.

Epidermal growth factor up-regulates CD44-dependent astrocytoma invasion in vitro.

CD44/hyaluronan interactions and epidermal growth factor (EGF) stimulation are both known to enhance tumour invasion in vitro. The frequent amplification of the EGF receptor (EGFR) in high-grade astrocytomas led to the examination of the hypothesis that CD44-dependent astrocytoma invasion is regulated by EGF. It has been shown that human astrocytoma cells express only the standard (haemopoietic) form of CD44 (CD44s) and that EGF up-regulates CD44 mRNA and protein in a time- and dose-dependent (10-100 ng/ml) manner. EGF stimulation did not result in induction of additional splice variants. No EGF-induced increase in CD44s was observed after treatment of cells with the wild-type EGFR tyrosine kinase inhibitor Tyrphostin AG1478 (30 nM). Up-regulation of CD44 by EGF is also prevented by the transcriptional inhibitor actinomycin D (5 microg/ml) and by blocking the MAP kinase (MAPK) pathway using the MEK inibitor U0126 (100 microM). CD44 up-regulation was associated with a 50% increase in invasion through hyaluronan-supplemented Matrigel(trade mark), which was abrogated by ligating CD44 with the specific antibody KM201. These results suggest that increased CD44 expression in response to EGF stimulation plays a significant role in astrocytoma invasion.

Compliant realignment of binding sites in muscle: transient behavior and mechanical tuning.

The presence of compliance in the lattice of filaments in muscle raises a number of concerns about how one accounts for force generation in the context of the cross-bridge cycle--binding site motions and coupling between cross-bridges confound more traditional analyses. To explore these issues, we developed a spatially explicit, mechanochemical model of skeletal muscle contraction. With a simple three-state model of the cross-bridge cycle, we used a Monte Carlo simulation to compute the instantaneous balance of forces throughout the filament lattice, accounting for both thin and thick filament distortions in response to cross-bridge forces. This approach is compared to more traditional mass action kinetic models (in the form of coupled partial differential equations) that assume filament inextensibility. We also monitored instantaneous force generation, ATP utilization, and the dynamics of the cross-bridge cycle in simulations of step changes in length and variations in shortening velocity. Three critical results emerge from our analyses: 1) there is a significant realignment of actin-binding sites in response to cross-bridge forces, 2) this realignment recruits additional cross-bridge binding, and 3) we predict mechanical behaviors that are consistent with experimental results for velocity and length transients. Binding site realignment depends on the relative compliance of the filament lattice and cross-bridges, and within the measured range of these parameters, gives rise to a sharply tuned peak for force generation. Such mechanical tuning at the molecular level is the result of mechanical coupling between individual cross-bridges, mediated by thick filament deformations, and the resultant realignment of binding sites on the thin filament.

Value of granulocyte colony stimulating factor in radiotherapy induced neutropenia: clinical and laboratory studies.

We report the effect of granulocyte colony stimulating factor (G-CSF) on neutropenia occurring during extended field radiotherapy in two groups of patients. The first group comprised 8 patients receiving craniospinal irradiation for a variety of central nervous system (CNS) neoplasms. None of these patients received cytotoxic chemotherapy. G-CSF was administered when the absolute neutrophil count (ANC) approached 1.5 x 10(9)/l. Neutropenia was promptly corrected in all cases, thereby avoiding unscheduled interruptions in radiotherapy. Following each G-CSF administration, ANC reached a peak on the following day and then declined steadily. Mean ANC rose from 1.33 x 10(9)/l on the day of G-CSF treatment to 7.07 x 10(9)/l the next day. Patients received 2-6 G-CSF injections during radiotherapy. Experiments were carried out in vitro to assess the risk of G-CSF causing increased CNS tumour cell proliferation. 11 human CNS tumour cultures (2 medulloblastomas, 2 primitive neuroectodermal tumours and 7 astrocytic tumours) were cultured in the presence of G-CSF at a range of concentrations up to 100 ng/ml. Their proliferation was compared with that of a G-CSF dependent murine leukemia cell line (NFS-60). None of the human tumour cultures demonstrated a significant increase in proliferation in response to G-CSF. 4 patients undergoing "mantle" type radiotherapy for Hodgkin's Disease or Non-Hodgkin's Lymphoma also received G-CSF treatment for neutropenia. All 4 had previously received cytotoxic chemotherapy. The number of G-CSF injections given per patient during radiotherapy ranged from 3-6. Mean ANC rose from 1.76 x 10(9)/l to 10.8 x 10(9)/l the next day. These results suggest that G-CSF is a reliable treatment for radiotherapy induced neutropenia and that an intermittent dosage schedule is effective.

New drug development in the United States, 1963 through 1984.

This study is the fifth in a series that documents changes in the status of drug development in the United States based on a survey of the pharmaceutical industry. For example, the study shows that, although there has been a recent increase in the number of new chemical entities being tested by US firms, an increasing proportion are acquired from outside the firm. Moreover, a growing number of acquired new chemical entities are coming from sources outside the United States, particularly Japan. These and other trends suggest an overall decline in research activity in the United States. At the same time, foreign firms are becoming more active, foretelling greater competition in the United States for both market share and research resources. The analyses also show a continued increase in synthesis-to-approval time, surpassing 13 years in the early 1980s, and rising success rates, reaching about 12% by the late 1970s.

New indications for already-approved drugs: time trends for the new drug application review phase.

For 55 individual drugs, the time required for approval of the original new drug application (NDA) was compared with the time required for approval of supplemental NDAs. The mean time required for the original NDAs was 22.2 +/- 18.1 months; the mean for review of all 46 follow-on indications was similar (19.1 +/- 17.9 months). Since subsequent applications do not ordinarily require new assessment of clinical safety or animal toxicity studies, one would expect the subsequent applications to be processed more expeditiously. Such is not the case. The negative implications of these facts are discussed.

The development of self-originated new drugs by Swiss pharmaceutical firms, 1960-1980.

This study of the development and regulation of self-originated new chemical entities (NCEs) in Switzerland between 1960 and 1980 is based on confidential data obtained through a survey of the three largest Swiss companies. Analyses of these data allowed description of the changes over time in (1) the number of NCEs introduced into clinical testing each year, (2) the time required to test and gain approval for a new drug, and (3) the success rate for NCEs, i.e., the proportion of those tested in man that received marketing approval. Because Swiss regulations may have an impact on these trends, the possible relationship between major regulatory events and changes in research activity is examined. Other factors such as regulatory requirements in major foreign markets and international economic influences are also considered.

New drug development by United States pharmaceutical firms with analyses of trends in the acquisition and origin of drug candidates, 1963-1979.

The average number of self-originated new chemical entities (NCEs) first tested in man by 39 United States-owned pharmaceutical firms in the 3-year period from 1977 to 1979 was 26 a year, approximately half the number investigated annually in the previous decade. Investigational New Drug (IND) Exemption filings on self-originated NCEs, but not those on acquired NCEs, were also comparatively low. Consequently, the contribution of self-originated NCEs to total IND filings fell from 81% in 1963 through 1975 to 68% in 1976 through 1979. (There was a similar decline, from 78% to 61%, in the proportion of compounds synthesized in the United States.) The relative increase in IND filings on acquired NCEs was greatest for smaller firms. By the late 1970s acquired NCEs accounted for almost 50% of the INDs filed by smaller firms, but only 10% to 25% of those filed by large and medium-sized firms. The importance of NCEs acquired from abroad has increased since the mid-1970s. The number of INDs filed on Japanese-originated NCEs rose from approximately one a year in 1963 through 1975 to an average of 3.5 a year in 1976 through 1979. Initial clinical testing of self-originated drugs abroad, which increased sharply in the early 1970s to reach a peak of 36% in 1976, declined to approximately 21% in 1977 through 1979. Self-originated drugs approved in 1977-1979 spent an average of 6 years in United States clinical testing and 2 in regulatory review, a total of 8 years from IND filing to NDA approval. The percentage of IND filings on self-originated NCEs that received New Drug Application (NDA) approval after 8 years or more was 9% overall, although ultimate success rates will be higher. There was a higher success rate for anti-infective drugs (17%) than for other pharmacologic categories (7%). For acquired NCEs, the overall approval rate was much larger (28%).

Abnormal enzyme phenotype (E1a E1f): normal response to succinylcholine.

The enzyme serum cholinesterase responsible for the hydrolysis of the muscle relaxant succinylcholine exists in the form of several variants. These may be identified in serum by using substances which inhibit their activity to different degrees. The heterozygote for the atypical and fluoride resistant enzymes (E1a E1f) is one of the phenotypes which has been reported to be sinsitive to succinylcholine. A case is described where succinylcholine given on two separate occasions did not induce apnoea in an individual phenotyped as E1a E1f by at least five methods of inhibition. This is the first reported example of such insensitivity to the drug in this phenotype. Temperature activities for the patient's serum over the range of 20 degrees C to 45 degrees C differed from that of an established E1a E1f phenotype used as a control. There was a progressive inactivation of the control serum at temperatures higher than 35 degrees C, as previously reported for this phenotype. Activity in the serum of the subject of this study did not exhibit the peak activity at 35 degrees C but continued to rise and probably reached a peak between 40 degrees C and 45 degrees C. The significance of these results in the context of current methods of phenotyping is discussed.

Aortocoronary bypass graft in dogs: late histological changes.

Late histological changes occurring in aortocoronary bypass vein grafts were studied by lignt and electron microscopy in three dogs killed one, two and three years after grafting. The changes consisted of intimal thickening due to a proliferation of modified smooth muscle cells (myointimal hyperplasia) and replacement of most of the medial smooth muscle by fibrocytes. Serial angiography in the dogs did not reveal progression of the intimal thickening after one month.