Single-step Exposure Research Articles

Simultaneously improving fabrication accuracy and interfacial bonding strength of multi-material projection stereolithography by multi-step exposure

ABSTRACT Multi-material additive manufacturing (MMAM) takes full advantage of the ability to arbitrarily place materials of additive manufacturing technology, enabling immense design freedom and functional print capabilities. Among MMAM technologies, projection stereolithography (PSL) exhibits a great balance of high resolution and fast printing speed. However, fabrication accuracy of multi-material PSL is hindered by large overcure used to strengthen interfacial bonding weakened by chemical affinity and material-exchange process. We present a novel multi-step exposure method for multi-material PSL process to overcome this shortcoming. Firstly, the whole layer is moderately exposed producing overcure of single-material PSL level to generate geometries. Then weakened interfaces are strengthened individually with additional steps of exposure. The multi-step exposure is integrated into the already efficient materials printing order of multi-material PSL process. Curing depth and overcure of photocurable resins are modeled and characterized. Exposure required to achieve sufficient interfacial bonding of single-material interfaces built through material-exchange process and multi-material interfaces with altering materials printing order is determined with tensile tests. Microfluidic channels are used to compare fabrication accuracy of traditional single-step exposure and our multi-step exposure method. This method can be widely applied in multi-material PSL to improve fabrication accuracy in a variety of applications including microfluidic devices.

Stitching-free 3D printing of millimeter-sized highly transparent spherical and aspherical optical components

We demonstrate the fabrication of optical elements on the millimeter scale by stitching-free 3D printing via two-photon polymerization, using a commercial microfabrication system (Nanoscribe GmbH). Previous limitations are overcome by the use of a large writing field objective as well as a novel high transparency resist. The printed optical components are free of stitching defects due to a single step exposure and exhibit an unpreceded glass-like appearance due to the low absorption of the resist material throughout the entire visible wavelength range. We print aspherical focusing lenses, characterize and optimize their shape fidelity, and find their optical performance close to the simulated optimum. For comparison with commercially available glass lenses we also fabricate spherical half-ball lenses of different sizes. The imaging quality of the lenses is very similar, underpinning the powerfulness of our fabrication strategy.

Osmotic tolerance of feline epididymal spermatozoa

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.

Single-Step Selection of Drug Resistant Acinetobacter baylyi ADP1 Mutants Reveals a Functional Redundancy in the Recruitment of Multidrug Efflux Systems

Members of the genus Acinetobacter have been the focus recent attention due to both their clinical significance and application to molecular biology. The soil commensal bacterium Acinetobacter baylyi ADP1 has been proposed as a model system for molecular and genetic studies, whereas in a clinical environment, Acinetobacter spp. are of increasing importance due to their propensity to cause serious and intractable systemic infections. Clinically, a major factor in the success of Acinetobacter spp. as opportunistic pathogens can be attributed to their ability to rapidly evolve resistance to common antimicrobial compounds. Whole genome sequencing of clinical and environmental Acinetobacter spp. isolates has revealed the presence of numerous multidrug transporters within the core and accessory genomes, suggesting that efflux is an important host defense response in this genus. In this work, we used the drug-susceptible organism A. baylyi ADP1 as a model for studies into the evolution of efflux mediated resistance in genus Acinetobacter, due to the high level of conservation of efflux determinants across four diverse Acinetobacter strains, including clinical isolates. A single exposure of therapeutic concentrations of chloramphenicol to populations of A. baylyi ADP1 cells produced five individual colonies displaying multidrug resistance. The major facilitator superfamily pump craA was upregulated in one mutant strain, whereas the resistance nodulation division pump adeJ was upregulated in the remaining four. Within the adeJ upregulated population, two different levels of adeJ mRNA transcription were observed, suggesting at least three separate mutations were selected after single-step exposure to chloramphenicol. In the craA upregulated strain, a T to G substitution 12 nt upstream of the craA translation initiation codon was observed. Subsequent mRNA stability analyses using this strain revealed that the half-life of mutant craA mRNA was significantly greater than that of wild-type craA mRNA.

Direct nanopatterning of 100 nm metal oxide periodic structures by Deep-UV immersion lithography

Deep-UV lithography using high-efficiency phase mask has been developed to print 100 nm period grating on sol-gel based thin layer. High efficiency phase mask has been designed to produce a high-contrast interferogram (periodic fringes) under water immersion conditions for 244 nm laser. The demonstration has been applied to a new developed immersion-compatible sol-gel layer. A sol-gel photoresist prepared from zirconium alkoxides caped with methacrylic acids was developed to achieve 50 nm resolution in a single step exposure. The nanostructures can be thermally annealed into ZrO(2). Such route considerably simplifies the process for elaborating nanopatterned surfaces of transition metal oxides, and opens new routes for integrating materials of interest for applications in the field of photocatalysis, photovoltaic, optics, photonics or microelectronics.

22.1: Binary Alignment Pattern Induced by Single Step Exposure of Laser Beam Polarization Interference

AbstractA new method of making binary alignment pattern through a single step exposure without using mask is proposed. This binary pattern is induced by polarization interference of laser beams at alignment surface. The line width is controlled by crossing angle between two input beams, which could be very small. Using azo‐dye as alignment material, which tends to align their long axis perpendicular to the activate light polarization direction, we obtain a binary pattern of 3 μm in pitch with laser beam crossing half angle at 3.48°.

Fmt Bypass in Pseudomonas aeruginosa Causes Induction of MexXY Efflux Pump Expression

The intrinsic resistance of P. aeruginosa PAO1 to the peptide deformylase inhibitor (PDF-I) LBM415 was mediated by the MexAB-OprM and MexXY-OprM efflux pumps, the latter of which was strongly induced by LBM415. Single-step exposure of PAO1 deleted for mexAB-oprM (therefore lacking both MexAB-OprM and MexXY-OprM functions) to PDF-Is selected for nfxB mutants, which express the MexCD-OprJ efflux pump, indicating that these compounds are also substrates for this pump. Selection of resistant mutants by use of levels of LBM415 greater than that accommodated by efflux yielded two additional groups of mutations, in the methionyl-tRNA(fmet) formyltransferase (fmt) and folD genes. Both mechanisms are known to impose an in vitro growth deficit (also observed here), presumably due to impairment of protein synthesis. We surmised that this inherent impairment of protein synthesis would upregulate expression of mexXY in a fashion similar to upregulation by LBM415 or by ribosome inhibitory compounds. Transcriptional profiling and/or mexX::lux promoter fusion analysis revealed that fmt and folD mutants were strongly upregulated for mexXY and another gene known to be required for upregulation of the pump, PA5471. Complementation of the fmt mutation in trans reversed this constitutive expression. This supports the notion that MexXY has a natural physiological function responding to impairment of ribosome function or protein synthesis and that fmt mutation (Fmt bypass) and folD mutation generate the intracellular mexXY-inducing signal.

Study of multi-step X-ray exposures to fabricate microstructure

Compared with single-step exposure of X-ray light source of high-power synchrotron radiation, lithographic process using multi-step exposures with a conformal mask has some advantages such as a thinner absorption layer, better heat dissipation, and shorter developing time. In this study, a conformal mask with an aurum (Au) absorber is fabricated using traditional ultraviolet (UV) lithography and Au electroplating process. For X-ray exposure, an analytical model of the absorption dosage (exposure dosage) along the exposed depth of x is developed. It can estimate accurately the time and dosage required for exposure. Based on the calculated dosage, 2-mm-thick PMMA (Polymethylmethacrylate) template with high-aspect ratio of 20 is fabricated by this multi-step exposures and developing process. With this template, nickel-cobalt (Ni–Co) alloy electroplating process is applied to fabricate a metallic mold with high hardness. For high-aspect-ratio microstructures, hardness and internal stress of the metallic mold by electroplating is a key factor to the quality. Experimentally, it is found that changing the ratio of Ni sulfamate to Co sulfamate can affect hardness and internal stress of the deposit. Though the hardness of the electroplated alloy is improved due to Co sulfamate, internal stress is induced at the same time. Thus, it is necessary to add a stress reducer to reduce internal stress. The currently result shows that the hardness of the mold of about 550 Hv with about 25 wt.% of Co and zero internal stress can be made successfully after adding a stress reducer. With this electroplating technique, the 2-mm-thick and 100-μm-wide microstructure with high hardness is fabricated.

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

P-175: Single Step Surface Alignment Patterning in Liquid Crystals Using Polarization Holography Exposure

We disclose a method creating two-dimensional patterned alignment layers using a single step exposure on a linear photopolymerizable polymer alignment layer. We demonstrate patterned circumferential and patterned radial alignment. These display configurations demonstrate continuously varying planar alignment patterns on the micrometer scale with low threshold voltage behavior < 2 V.

Feasibility of a Nylon-Mesh Holder for Vitrification of Bovine Germinal Vesicle Oocytes in Subsequent Production of Viable Blastocysts1

To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.

Suppressive Effect of 1α,25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functional Activities and Chemotaxis

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active metabolite of vitamin D(3), on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)(2)D(3) prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)(2)D(3)-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)(2)D(3). Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)(2)D(3) is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)(2)D(3)-mediated immunomodulation.

Multimode transflective liquid crystal display with a single cell gap using a self-masking process of photoalignment

We demonstrate a transflective liquid crystal display (LCD), doped with a chiral agent to produce a low helical twisting power, in a multimode configuration consisting of the homogeneous alignment and the hybrid alignment. The multimode transflective LCD was fabricated by a single-step exposure of the UV light through an array of metal reflectors used as an amplitude photomask which gives an alternating homogeneous and homeotropic LC geometry. This single-step UV exposure produces no cell gap variations. In our configuration, the electro-optical disparity between the transmissive region and the reflective region was found to be significantly reduced by the low helical twisting power of the chiral dopant.

81IN VITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF BOVINE IMMATURE OOCYTES CRYOPRESERVED BY VITRIFICATION WITH STEPWISE EXPOSURE USING A NYLON MESH

Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2–5mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3M sucrose (EFS40) or 0.3M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5cm2), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P&amp;lt;0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P&amp;lt;0.05). Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.

Single-Step Microstructure Patterning in Titania-Doped Sol-Gel Glassy Thin Films with Electron Beams

ABSTRACTTo achieve a simplified microstructure patterning process, patterning in thin films with a singlestep exposure to UV-light or E-beam process for fabrication of integrated optical waveguides and components is of great interest in optics, because the complicated structure developing stage by wet chemical or plasma etching is not required. In this paper, we describe our study on the microstructure patterning in TiO2-doped hybrid glassy polymer thin films by irradiating with focused E-beam. An anhydrous sol-gel process based on hydrolysis and condensation of metal alkoxides with boric acid under non-hydrous conditions was applied to homogeneously incorporating TiO2into silica network, and transparent glassy TiO2-doped thin films with varied TiO2concentrations (up to 40 mol%) were prepared by spin coating the hybrid polymer onto fused silica substrates and silicon wafers. The TiO2-doped hybrid material showed a low OH absorption, low optical losses at the telecommunication windows (&lt;0.40 dB/cm at 1550 nm and &lt;0.20 dB/cm at 1310 nm respectively), good thermo-optical linearity and good thermal stability up to 200 °C. Single-step direct surface patterning in the TiO2-doped hybrid glassy films with focused electron beams irradiation showed that fine structure with resolution at submicron level can be fabricated. As the process does not need a developing (etching) process, the profiles of the lines fabricated by the E-beam direct writing showed few distortion. The high precise patterning microstructure with submicron feature sizes in the TiO2-doped hybrid glassy thin films may provide potentials for applications to the fabrication of optical gratings.

Glycopeptide resistance in coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) were the first organisms in which acquired glycopeptide resistance was recognized. Ever since the early reports, it has been apparent that resistance to teicoplanin is more common than that to vancomycin and that resistance occurs mostly in species such as Staphylococcus haemolyticus and Staphylococcus epidermidis. The minimum inhibitory concentrations (MICs) of teicoplanin for CNS usually fall over a wide range, and, especially in some methicillin-resistant isolates of the two above-mentioned species, they can reach and even exceed the resistance breakpoint, whereas vancomycin MICs tend to remain more stable over a narrower range within the limits of susceptibility. CNS strains intermediately susceptible and even resistant not only to teicoplanin but also to vancomycin have, however, been isolated, most frequently from patients subjected to prolonged glycopeptide treatment. Laboratory detection of glycopeptide-resistant CNS may be problematic, mainly because susceptibility tests, particularly those for teicoplanin, are influenced by various technical factors, and agar diffusion tests may yield false susceptibility data. In studies with experimental glycopeptides, some molecules have exhibited improved in vitro activity compared with teicoplanin and vancomycin, but these encouraging microbiological findings have not usually been followed by in vivo trials. Stepwise and single-step exposure to teicoplanin and vancomycin has allowed stable clones for which glycopeptide MICs are increased to be obtained from susceptible CNS strains, particularly strains of Staphylococcus haemolyticus and Staphylococcus epidermidis. In these studies, resistance to teicoplanin was generally easier to obtain than resistance to vancomycin, and the levels of teicoplanin resistance were higher. Population studies have demonstrated the usually heterogeneous nature of glycopeptide resistance in CNS. Although glycopeptide-resistant CNS have been shown to differ in several features from their glycopeptide-susceptible counterparts, the exact mechanism of staphylococcal glycopeptide resistance remains unknown.

Assessment of the effect of dimethyl sulfoxide on the survival of cryopreserved mouse morulae by single step exposure and rapid freezing

Assessment of the effect of dimethyl sulfoxide on the survival of cryopreserved mouse morulae by single step exposure and rapid freezing

Low-temperature chemical vapour deposition processes and dielectrics for microelectronic circuit manufacturing at IBM : D. R. Cote, S. V. Nguyen, W. J. Cote, S. L. Pennington, A. K. Stamper and D. V. Podlesnik. IBM J. Res. Develop., 1995, 39(4), 437

Compared with single-step exposure of X-ray light source of high-power synchrotron radiation, lithographic process using multi-step exposures with a conformal mask has some advantages such as a thinner absorption layer, better heat dissipation, and shorter developing time. In this study, a conformal mask with an aurum (Au) absorber is fabricated using traditional ultraviolet (UV) lithography and Au electroplating process. For X-ray exposure, an analytical model of the absorption dosage (exposure dosage) along the exposed depth of x is developed. It can estimate accurately the time and dosage required for exposure. Based on the calculated dosage, 2-mm-thick PMMA (Polymethylmethacrylate) template with high-aspect ratio of 20 is fabricated by this multi-step exposures and developing process. With this template, nickel-cobalt (Ni–Co) alloy electroplating process is applied to fabricate a metallic mold with high hardness. For high-aspect-ratio microstructures, hardness and internal stress of the metallic mold by electroplating is a key factor to the quality. Experimentally, it is found that changing the ratio of Ni sulfamate to Co sulfamate can affect hardness and internal stress of the deposit. Though the hardness of the electroplated alloy is improved due to Co sulfamate, internal stress is induced at the same time. Thus, it is necessary to add a stress reducer to reduce internal stress. The currently result shows that the hardness of the mold of about 550 Hv with about 25 wt.% of Co and zero internal stress can be made successfully after adding a stress reducer. With this electroplating technique, the 2-mm-thick and 100-μm-wide microstructure with high hardness is fabricated.

Glyphosate Tolerance in Tobacco (Nicotiana tabacum L.)

A glyphosate-tolerant tobacco cell line, Nicotiana tabacum L. Indiana (I7), was selected from the glyphosate-sensitive Wisconsin 38 (W38) line through a single step exposure to the herbicide. Tolerance and growth characteristics of I7 cells were the same for cells maintained for more than 1 year in the presence or absence of glyphosate. Glyphosate tolerance levels were constant through the growth cycle. Tolerance is not due to reduced uptake of glyphosate. Shikimate levels in I7 and W38 cells maintained in glyphosate-free medium were similar, whereas W38 cells accumulated 46 times more shikimate than I7 cells, when cells of both lines were exposed to the herbicide. Glyphosate treatment caused increased levels of aromatic amino acids in W38 cells and slightly lower levels in I7 cells. Specific activities of dehydroquinate synthase, shikimate dehydrogenase, and shikimate kinase were similar in the two cell types, whereas DAHP synthase and EPSP synthase specific activities were elevated in I7 cells. Plants regenerated from I7 cells retained tolerance to glyphosate.