Determination of CEBPA mutations by next generation sequencing in pediatric acute leukemia.

OBJECTIVES: The CCAAT/enhancer-binding protein-alpha (CEBPA) is lineage-specific transcription factor in the hematopoietic system. In this study, we aimed on the clinical features and the prognostic significance associated with CEBPA mutations in 30 pediatric patients with acute leukemia. METHODS: In addition, the association between found variants and mutations of Ten-Eleven-Translocation 2 (TET2), Kirsten rat sarcoma viral oncogene homolog (KRAS), and Casitas B-cell lymphoma (CBL), FLT3 (Fms-Related Tyrosine Kinase), JAK2 (Januse Kinase-2) and Nucleophosmin 1 (NPM1) were analyzed, which are important prognostic risk factors for pediatric acute leukemia patients. The entire CEBPA coding region was screened using the NGS method. RESULTS: CEBPA mutations were detected in 16 (53.3 %) of 30 patients. In total, ten distinct of nucleotide changes were identified in 30 patients, including 6 novel and 4 known mutations by sequencing the entire CEBPA gene. We found 6 frame shift mutations, 1 missense mutation, 3 synonymous variants. The most common mutation was the c.487del G resulting p.Glu163Ser in 5 cases. Three patients carried CEBPA double mutations. CONCLUSION: The detected variants in this article seemed to be the first screening results of genes studied by NGS in pediatric acute leukemia patients. Our results also showed some degree of association between FLT3-ITD, TET2, KRAS, CBL and CEBPA mutations (Tab. 4, Fig. 1, Ref. 24).

Lepidoptera (Crambidae, Noctuidae, and Pyralidae) Injury to Corn Containing Single and Pyramided Bt Traits, and Blended or Block Refuge, in the Southern United States.

Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae); corn earworm, Helicoverpa zea Boddie (Lepidoptera: Noctuidae); southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae); sugarcane borer, Diatraea saccharalis F. (Lepidoptera: Crambidae); and lesser cornstalk borer, Elasmopalpus lignosellus Zeller (Lepidoptera: Pyralidae), are lepidopteran pests of corn, Zea mays L., in the southern United States. Blended refuge for transgenic plants expressing the insecticidal protein derivative from Bacillus thuringiensis (Bt) has recently been approved as an alternative resistance management strategy in the northern United States. We conducted a two-year study with 39 experiments across 12 states in the southern United States to evaluate plant injury from these five species of Lepidoptera to corn expressing Cry1F and Cry1Ab, as both single and pyramided traits, a pyramid of Cry1Ab×Vip3Aa20, and a pyramid of Cry1F×Cry1Ab plus non-Bt in a blended refuge. Leaf injury and kernel damage from corn earworm and fall armyworm, and stalking tunneling by southwestern corn borer, were similar in Cry1F×Cry1Ab plants compared with the Cry1F×Cry1Ab plus non-Bt blended refuge averaged across five-plant clusters. When measured on an individual plant basis, leaf injury, kernel damage, stalk tunneling (southwestern corn borer), and dead or injured plants (lesser cornstalk borer) were greater in the blended non-Bt refuge plants compared to Cry1F×Cry1Ab plants in the non-Bt and pyramided Cry1F×Cry1Ab blended refuge treatment. When non-Bt blended refuge plants were compared to a structured refuge of non-Bt plants, no significant difference was detected in leaf injury, kernel damage, or stalk tunneling (southwestern corn borer). Plant stands in the non-Bt and pyramided Cry1F×Cry1Ab blended refuge treatment had more stalk tunneling from sugarcane borer and plant death from lesser cornstalk borer compared to a pyramided Cry1F×Cry1Ab structured refuge treatment. Hybrid plants containing Cry1F×Cry1Ab within the pyramided Cry1F×Cry1Ab blended refuge treatment had significantly less kernel damage than non-Bt structured refuge treatments. Both single and pyramided Bt traits were effective against southwestern corn borer, sugarcane borer, and lesser cornstalk borer.

Prognostic and predictive significance of GITR in metastatic renal cell carcinoma.

OBJECTIVE: Renal cell carcinoma (RCC) has gradually increased in recent years. There have been significant developments in metastatic RCC in recent years with the introduction of immune control point inhibitors. Glucocorticoid-induced tumor necrosis factor (TNF) receptor-related protein (GITR) is a co-stimulatory molecule and is seen in the highest amounts in activated CD4+ T lymphocytes and CD8+ T lymphocytes, forkhead box protein 3 (FOXP3) positive regulatory T cells (Treg). GITR leads to an increase in interleukin (IL)-2 and CD25 and Interferon Gamma. It shows an anti-tumoural effect by inhibiting the suppressive functions of FOXP3+ regulatory cells (Treg). Therefore, we aimed to evaluate the prognostic and predictive effect of GITR, tumor-infiltrating lymphocytes (CD4+CD8) (TIL), and FOXP3 in patients with metastatic RCC. PATIENTS AND METHODS: Patients diagnosed with pathologically confirmed metastatic renal cancer between 2016 and 2021 were included in our study. Clinicopathological features and some laboratory tests were recorded. GITR, CD4, CD8, and FOXP3 were evaluated by immunohistochemistry (IHC) from biopsies or nephrectomy material and recorded. RESULTS: The study included 41 patients. The median progression-free survival (PFS) was 10.5 months, and the median overall survival (OS) was 13.9 months. Median PFS was 7.9 months for the GITR-low group and 18.9 months for the GITR-high group. Median PFS was statistically significant and longer for the GITR-high group than the GITR-low group (p=0.003). When patients who received nivolumab in the 2nd line were evaluated, median PFS was found to be 5.7 months in the GITR-low group and 15.7 months in the GITR-high group. Median PFS was statistically significantly higher in the GITR-high group than in the GITR-low group (p=0.026). CONCLUSIONS: In patients with metastatic RCC, higher GITR was associated with better PFS. At the same time, in patients using nivolumab, better PFS was seen in the GITR high group. If supported by prospective studies, GITR can be used as both a prognostic and predictive marker.

Ultrasound as a diagnostic tool to differentiate acute from chronic renal failure.

AIM: Renal ultrasound (US) is the most appropriate method for imaging renal failure; however, considerable overlap in renal size and renal echogenicity exists between normally and abnormally functioning kidneys. We compared the sonographic features of kidneys in patients with renal failure to investigate the potential role of renal US to distinguish acute from chronic renal failure and assessed the diagnostic role of body surface area-corrected renal length compared to measured renal length. MATERIALS AND METHODS: We included 127 consecutive patients with serum creatinine levels higher than 3 mg/dl and 33 healthy volunteers. The subjects with acute renal failure (ARF) and chronic renal failure (CRF) were compared for renal length, parenchymal thickness, parenchymal echogenicity, distinctness of the corticomedullary junction, and the presence of stones and cysts. RESULTS: No significant differences in age, serum albumin, creatinine, weight, height, or gender distribution were found between patients with ARF and those with CRF, except in serum hemoglobin. The right and left kidney parenchymal thickness and renal length were significantly greater in ARF patients than in those with CRF (p < 0.0001). The mean parenchymal thickness and renal length were similar in ARF patients and the control group. Grade I hyperechogenicity was the most common finding during sonography. CONCLUSIONS: Renal length, parenchymal thickness, and echogenicity differed significantly between patients with acute and chronic renal failure. A renal US examination is still the most appropriate method for imaging renal failure and should be combined with other tests to distinguish acute from chronic renal failure.

Signal-mediated nuclear transport in proliferating and growth-arrested BALB/c 3T3 cells.

Mediated transport across the nuclear envelope was investigated in proliferating and growth-arrested (confluent or serum starved) BALB/c 3T3 cells by analyzing the nuclear uptake of nucleoplasmin-coated colloidal gold after injection into the cytoplasm. Compared with proliferating cells the nuclear uptake of large gold particles (110-270 A in diameter, including the protein coat) decreased 5.5-, 33-, and 78-fold, respectively, in 10-, 14-17-, and 21-d-old confluent cultures; however, the relative uptake of small particles (total diameter 50-80 A) did not decrease with increasing age of the cells. This finding suggests that essentially all pores remain functional in confluent populations, but that most pores lose their capacity to transport large particles. By injecting intermediate-sized gold particles, the functional diameters of the transport channels in the downgraded pores were estimated to be approximately to 130 and 110 A, in 14-17- and 21-d-old cultures, respectively. In proliferating cells, the transport channels have a functional diameter of approximately 230 A. The mean diameters of the pores (membrane-to-membrane distance) in proliferating and confluent cells (728 and 712 A, respectively) were significantly different at the 10%, but not the 5%, level. No differences in pore density (pore per unit length of membrane) were detected. Serum-deprived cells (7-8 d in 1% serum or 4 d in 0.5% serum) also showed a significant decrease in the nuclear uptake of large, but not small, gold particles. Thus, the permeability effects are not simply a function of high cell density but appear to be growth related. The possible functional significance of these findings is discussed.

The permeability of the nuclear envelope in dividing and nondividing cell cultures.

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.

Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175.

INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.

Anticardiolipin antibodies in hemodialysis patients with hepatitis C and their role in fistula failure.

BACKGROUND/AIMS: Several conflicting results are presently reported regarding raised IgG and IgM-anticardiolipin antibodies (ACA) titers in hemodialysis (HD) patients and their role in vascular access dysfunction. We aimed to determine the prevalence of both IgM and IgG-ACA titers and to analyze retrospectively their role in primary and secondary arteriovenous fistula (AVF) failure in a homogeneous group of HD patients with chronic hepatitis C. METHODS: This study included 103 adults on maintenance hemodialysis with chronic hepatitis C infection. All participants had blood samples drawn predialysis and after an overnight fast. Analysis included biochemistry, IgG and IgM ACA, Anti-HCV, HBsAg, serum HCV RNA and HCV genotyping. RESULTS: The prevalence of IgG-ACA was 14.6% (15/103). No patient had a positive value of the IgM-ACA test. HCV replication was detected in 52 of 76 patients. The most common HCV genotype was genotype 1 (90%). The percentage of females was higher in ACA(+) group (p = 0.038). There were no significant differences between subjects with and without ACA-IgG regarding other parameters studied. No difference in regard to AVF survival was detected between ACA(+) and ACA(-) groups (p > 0.05). CONCLUSION: We found no significant differences in primary or secondary AVF failure between patients with elevated and normal ACA. Therefore, we conclude that AVFF may be caused by factors other than ACA in these patients. More prospective studies are needed to confirm this observation.

Tarnished plant bug (Hemiptera: Miridae) thresholds and sampling comparisons for flowering cotton in the midsouthern United States.

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), has become the primary target of foliar insecticides in cotton, Gossypium hirsutum L., throughout the Midsouth over the past several years. This prompted a reevaluation of existing action thresholds for flowering cotton under current production practices and economics. A trial was conducted at 19 locations throughout the Midsouth during 2006 and 2007. Threshold treatments ranged from a weekly automatic insecticide application to a very high threshold of 10 tarnished plant bugs per 1.5 row-m on a black drop cloth. Individually, all locations reached the lowest threshold, and eight locations had a significant yield loss from tarnished plant bugs. Across all locations, lint yield decreased 0.85 to 1.72% for each threshold increase of one tarnished plant bug per 1.5 row-m. Yield loss was most closely correlated to pest density during the latter half of the flowering period. The relationship between plant bug density or damage and yield was similar for drop cloth, sweep net, and dirty square sampling methods, but the correlations among these sampling methods were not high. Incorporating actual insecticide application data from the trial and average production and economic factors for Midsouth cotton, the economic threshold, if monitoring once per week, should be between 1.6 and 2.6 tarnished plant bugs per 1.5 row-m during the flowering period. More frequent monitoring or situations where insecticide applications are more efficacious may alter this threshold.

Tarnished plant bug (Hemiptera: Miridae) thresholds for cotton before bloom in the Midsouth of the United States.

Insecticide applications to control tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), during cotton, Gossypium hirsutum L., bud formation are common throughout the Midsouth of the United States. Cultivation practices and the pest complex have changed since action thresholds were established for this pest. A trial was conducted at 33 locations over 3 yr throughout the Midsouth to evaluate tarnished plant bug damage to cotton during the prebloom period. There was no consistent yield response to action thresholds, but average tarnished plant bug density and average square loss were both significant factors impacting lint yield. Based on the yield responses and application frequency of the various action thresholds, the best economic scenario occurred when tarnished plant bug density during the prebloom period averaged eight per 100 sweeps and square retention averaged 90%. The action thresholds required to achieve these averages are expected to be higher than these levels because pest pressure is not normally constant during the prebloom period. When insecticides are required, an application interval shorter than one week may be needed to obtain satisfactory control.

Comparison of cone-beam computed tomography and panoramic radiography for mandibular morphometry.

BACKGROUND: The aim of this study was to compare the morphological differences in the mandible between patients with six age groups and to detect the correlation between these parameters on panoramic radiography (PR) and cone-beam computed tomography (CBCT). MATERIALS AND METHODS: A total of 121 subjects (50 males and 71 females) were included in the study and were divided into six age groups (10-19, 20-29, 30-39, 40-49, 50-59, and 60-69) on the basis of the chronological age. CBCT and PR methods were used to record the mandibular measurements for the same 121 patients. Differences between male and female mandibular morphometric measurements, between right and left side measurements, and differences in age subgroups compared by using independent samples t-test, paired samples t-test, and one-way ANOVA test, respectively. P < 0.05 value was considered statistically significant for all analysis. RESULTS: Males mostly have higher mandibular measurement values. There were statistically significant differences between CBCT and PR measurements (p < 0.05). PR mostly showed higher values than CBCT measurements. CONCLUSIONS: Based on the fact that PRs showing significant differences from CBCT in the morphometric measurements made on mandible, it is recommended that forensic doctors and anthropologists consider this information in their age and gender prediction studies.

Does the direction of arterial needle in AV fistula cannulation affect dialysis adequacy?

AIM: The direction of arterial access needles in fistulas and grafts has been a subject of some controversy and there is no study comparing the results either direction of arterial needle placement in cannulation of arteriovenous fistula. We compared mean urea reduction rate (URR) and Kt/V in the same HD patients when dialyzed via antegrade or retrograde arterial needle cannulation. MATERIALS AND METHODS: This was a study involving 22 adults on maintenance hemodialysis for more than 6 months. Doppler US examinations of arteriovenous fistula were performed in all subjects. Pre-dialysis and post-dialysis blood samples were obtained at the patient's midweek HD treatment 4 times a month for each direction. Arterial needle was placed in retrograde direction for the first month. On the second month, the direction of arterial needle was converted to antegrade. Means were compared by paired t-test. RESULTS: Mean URR and eKt/Vof retrograde cannulation were 74.2+/-7.2% and 1.57+/-0.33. The results were indifferent statistically from those of antegrade cannulation (73.0+/-8.7% and 1.57+/-0.35 (p=0.123)). Mean fistula blood flow was 931 +/- 483 ml/min. No cannulation complication was observed during the study period for both directions. CONCLUSIONS: Both antegrade and retrograde arterial needle placement may be preferred according to center experience without concern of HD adequacy. Longterm outcomes of antegrade and retrograde arterial needle placement such as AVF failure, thrombosis, and stenosis warrant further studies.

Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

Cytochemistry and ultrastructural morphometry of cultured HL60 myeloid leukemia cells.

Cultured human myeloid leukemia (HL60) cells were characterized using ultrastructural cytochemical methods and differences identified when cells were compared for low (17 to 47), middle (69 to 100), and high (214 to 244) passages or to normal promyelocytes aspirated from bone marrow. Endoplasmic reticulum and transition structures (pre-Golgi compartment) of HL60 cells stained positively for peroxidase using diaminobenzidine but stained sparsely for reducing groups with osmium-zinc iodide. Staining of Golgi elements was relatively indistinct with diaminobenzidine and strong with osmium-zinc iodide, in comparison to freshly harvested promyelocytes which have intense diaminobenzidine and osmium-zinc iodide staining of the pre-Golgi and Golgi compartments. Cytoplasmic polyribosomes were more numerous in middle and high passage cells, whereas dilatation of endoplasmic reticulum was less prominent in these cells. The mean granule size was significantly increased in low passage cells, and staining of peroxidase was more prominent by light and electron microscopy when compared to high passage cells. Cytoplasmic granules demonstrated strong complex carbohydrate staining, indicating a lack of granule maturation in HL60 cells. Terminally differentiated myeloid cells were more frequent in low passage samples, and some neutrophil granule maturation appeared to occur within these cells, whereas all eosinophil granules consistently remained immature with intense complex carbohydrate staining and lack of crystalloid formation. These studies demonstrate significant differences between HL60 cells and normal promyelocytes, and also passage-dependent maturational differences in HL60 cells. These differences should be considered in evaluating parameters of cell growth and maturation and in the biochemical and enzymatic characterization of these cells.

Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.

NTF2 monomer-dimer equilibrium.

Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 microM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.

Enzyme-retting of flax and characterization of processed fibers.

Enzyme-retting formulations consisting of Viscozyme L, a pectinase-rich commercial enzyme product, and ethylenediaminetetraacetic acid (EDTA) were tested on Ariane fiber flax and North Dakota seed flax straw residue. Flax stems that were crimped to disrupt the outer layers were soaked with various proportions of Viscozyme-EDTA solutions, retted, and then cleaned and cottonized with commercial processing equipment. Fiber properties were determined and crude test yarns were made of raw and Shirley cleaned flax fibers and cotton in various blend levels. Cleaned fibers were obtained from both seed and fiber flax types, but with variations due to treatment. Retting formulations produced fibers having different properties, with enzyme levels of 0.3% (v/v as supplied) giving finer but weaker fibers than 0.05% regardless of EDTA level. Experimental yarns of blended flax and cotton fibers varied in mass coefficient of variation, single end strength, and nep imperfections due to sample and formulation. With cost and fiber and yarn quality as criteria, results established a range in the amounts of components comprising retting formulations as a basis for further studies to optimize enzyme-retting formulations for flax. Under conditions examined herein, Viscozyme L at 0.3% (v/v) plus 25 mM EDTA produced the best test yarns and, therefore, established a base for future studies to develop commercial-grade, short staple flax fibers for use in textiles.

Chemical composition of components comprising bast tissue in flax.

Components from the bast region of flax (Linum usitatissium) were removed by hand from seed flax and fiber flax and analyzed by gas chromatography and mass spectrometry. Stems soaked in water were separated by hand into an outer layer, which consisted of epidermis with cuticle and parenchyma cells, and fiber bundles. Each of the two fractions were evaluated for chemical markers that could be used to predict the degree of retting and possibly fiber quality. The outer layer was subsequently treated with a mixed enzyme preparation to remove the carbohydrate portion, thus providing another fraction consisting primarily of cuticle. Four main constituent groups were investigated: dihydroxy fatty acids, long-chain fatty acids and alcohols, sterols, and aromatics. Long-chain fatty acids and alcohols located in the outer layer accounted for 80-92% of the total found in both fractions. Aromatics and sterols in the outer layer accounted for 29--72 and 27--67%, respectively, of the total and do not appear to be a reliable marker for the degree of retting. The best markers for retting were the dihydroxy fatty acids, of which 98--99% were accounted for in the outer layer. The main dihydroxy fatty acids were a mixture of 8,16- and 9,16-dihydroxyhexadecanoic acids and represented 87--89% of the total dihydroxy fatty acids measured. As a constituent of cuticle, this compound may serve as an excellent marker for indicating the degree of retting as well as a possible marker for fiber quality because this compound is almost always exclusively associated with the outer layer and not the fiber.

Chemical and spectroscopic analysis of lignin in isolated flax fibers.

The chemistry of pure flax fibers, free of contaminating nonfiber components, has not been determined. Fibers from the center sections of the stem of seed and fiber flax (Linum usitatissium L.), which had been retted after soaking in water and removal of the epidermis by hand, underwent chemical and spectroscopic analysis. Wet chemical analysis showed only trace indications of aromatics and no long chain fatty acids or alcohols in fibers. Pyrolysis mass spectroscopy (PyMS) and pyrolysis gas chromatography mass spectrometry (PyGCMS) showed only trace amounts of aromatic constituents that could be attributed to the presence of lignin. Mid-infrared (Mid-IR) and Raman spectroscopy of these fibers showed no aromatic compounds present. This study suggests that earlier work reporting the presence of lignin ranging from 1 to 4% may be the result of residual shive or epidermis/cuticle material remaining after the retting process which may be responsible for the favorable properties desired by the composites industry.

Plant factors influencing enzyme retting of fiber and seed flax.

Retting, which is the microbial activity through which bast fibers are released from nonfiber tissues, is the limiting factor in flax processing. The objective of this work is to identify chemical and structural characteristics in a variety of fiber and seed flax types that influence enzyme retting in a recently developed method. Analyses of flax retted in a series of tests, including two enzyme rettings in some cases, indicated that lignin did not limit the separation of fibers from shive and showed that pectinases in enzyme-retting mixtures could ret fiber and seed flax. However, mature stems, such as that in flax produced for seed, had greater amounts of cutin and wax in the cleaned fiber product, suggesting that the cuticle could be a greater antiquality factor in seed versus fiber flax. With seed flax, the fraction of finer fibers produced during retting was significantly lower than with fiber flax. Results indicated that enzyme retting could be used to obtain flax fibers from seed flax stem residues and add value to this agricultural material.