Evaluation the Presence of SERPINA5 (Exon 3) and FTO rs9939609 Polymorphisms in Papillary Thyroid Cancer Patients.

BACKGROUND: A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC) globally. Here, we examined the presence of genetic variations within coding exon 3 of SERPINA5 gene and FTO rs9939609 polymorphism in Iranian PTC patients. METHODS: A total of 122 patients (42 cases for SERPINA5 and 80 cases for FTO gene) and 120 healthy subjects (40 subjects or SERPINA5 and 80 subjects for FTO gene) were recruited. The genetic variation within coding exon 3 of SERPINA5 gene was evaluated by reaction-single-strand conformation polymorphism (PCR-SSCP) and FTO rs9939609 polymorphism was evaluated by RFLP-PCR assay. RESULTS: The PCR-SSCP technique detected two rs6115G>A and rs6112T>C genetic variations within coding exon 3 of SERPINA5 gene and approved also by direct sequencing. For rs6112T>C polymorphism seven patients was heterozygous and for rs6115G>A seven PTC patients were heterozygous and two patients were homozygous. CONCLUSION: This study indicated that SERPINA5 rs6115G>A and rs6112T>C polymorphisms might be a novel susceptibility locus for PTC in Iranian patients. However, our findings do not support an association between FTO rs9939609 polymorphism and PTC risk.

Inactivating mutations of class II transactivator (CIITA) gene in gastric and colorectal cancers.

Expression of MHC class II, which is important against cancer immunity, depends on the transcactivator CIITA. These data suggest a possibility that CIITA gene might be inactivated in cancers. In this study, we studied inactivating mutation status of CIITA gene in gastric (GC) and colorectal (CRC) cancers by analyzing the C7 repeat in the CIITA (exon 11) gene. We found frameshift mutations in 3 GCs and 6 CRCs in cancers with high microsatellite instability (MSI-H) (9/113), but not in those with microsatellite-stable (MSS) (0/90) (P = 0.004). They were all deletion or duplication of one base in the C7 repeat that would result in truncation of amino acid synthesis. Immune therapy is now a major option in cancer therapy and our results on the genetic alterations of MHC II-related CTIITA in MSH-H GC and CRC might provide useful information for the treatment of MSI-H cancers.

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species.

Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E+2-13/M+2-13Hr158 generated PCR products for just H. harpax, while E+3-14/M+3-2HhHr151 and E+2-13/M+2-13Hh150 generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E+2-13/M+2-13Hr158, E+3-14/M+3-2HhHr151, and E+2-13/M+2-13Hh150 were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.

Resolving human DNA mixtures with F-108 polymer and capillary electrophoresis single-strand conformational polymorphism analysis.

Current technologies have increased the sensitivity for analyzing forensic DNA samples, especially those considered "touch samples." Because of this, there has been an increase in the number of forensic mixtures-two or more contributors within a single sample-submitted to the crime laboratories. Therefore, the need to resolve these mixtures has increased as well. Several technologies are currently utilized, but many of them are time consuming and do not resolve the entire profile. Therefore, CE-Single-Strand Conformational Polymorphisms coupled with the Pluronic F-108 polymer was assessed for its ability to resolve human forensic mixtures. This technique has been able to detect sequence variation, such as single nucleotide polymorphism in short tandem repeat loci, such as D7S820 and vWA. Samples were first analyzed with the Performance Optimized Polymer-7, and mixtures created from samples that shared alleles. These samples were sequenced to detect single base-pair mutations and evaluated with the F-108 and CE-Single Strand Conformational Polymorphism analysis. Results from this study indicated the method would serve as a valuable screening tool to detect base sequence variation between individuals when they share alleles in a mixture and before using Massive Parallel Sequencing technology to distinguish which bases differ.

Identification of the Ovine Keratin-Associated Protein 2-1 Gene and Its Sequence Variation in Four Chinese Sheep Breeds.

Keratin-associated proteins are important components of wool fibers. The gene encoding the high-sulfur keratin-associated protein 2-1 has been described in humans, but it has not been described in sheep. A basic local alignment search tool nucleotide search of the Ovine Genome Assembly version 4.0 using a human keratin-associated protein 2-1 gene sequence revealed a 399-base pair open reading frame, which was clustered among nine previously identified keratin-associated protein genes on chromosome 11. Polymerase chain reaction-single strand conformation polymorphism analysis revealed four different banding patterns, with these representing four different sequences (A-D) in Chinese sheep breeds. These sequences had the highest similarity to human keratin-associated protein 2-1 gene, suggesting that they represent variants of ovine keratin-associated protein 2-1 gene. Nine single nucleotide variations were detected in the gene, including one non-synonymous nucleotide substitution. Differences in variant frequencies between fine-wool sheep breeds and coarse-wool sheep breeds were detected. The gene was found to be expressed in various tissues, with the highest expression level in skin, and moderate expression levels in heart and lung tissue. These results reveal that the ovine keratin-associated protein 2-1 gene is variable and suggest the gene might affect variation in mean fiber diameter.

Variation in the yak lipin-1 gene and its association with milk traits.

The aim of this research was to identify variation in the yak lipin-1 gene (LPIN1) and determine whether this variation affects milk traits. PCR-single stranded conformational polymorphism (PCR-SSCP) analysis was used to detect variation in the 5' untranslated region of LPIN1 in 500 yaks from four populations: Tianzhu white yaks, Qinghai yaks, wild × domestic-cross yaks and Gannan yaks. Four unique PCR-SSCP patterns, representing four different DNA sequence variants (named A, B, C and D), were observed. These contained six single nucleotide polymorphisms. Female Gannan yaks with BC genotype produced milk with a higher fat content (P < 0.001) and total milk solids (P < 0.001), than those with the AA, AB and BB genotypes. These results would suggest that LPIN1 is having an effect on yak milk fat synthesis.

Association of growth performance and body conformational traits with BMP4 gene variation in Barki lambs.

The objective of this study was to test the association of the variation in a 360 bp region in exon 2 of the ovine bone morphogenetic protein 4 (BMP4) gene with growth performance (birth weight, pre-weaning average daily gain, weaning weight, post-weaning average daily gain and marketing weight) and body conformational traits (height at withers, height at hips, body length, heart girth, thigh circumference, body mass index, skeletal muscle index, body index and relative body index) in 242 Barki lambs using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). Two variants (A and B) and three genotypes (AA, AB and BB) were detected. The BMP4 genotype significantly affected (p < .05 or p < .01) post-weaning daily gain, marketing weight, height at hips, thigh circumference, body mass index and skeletal muscle index. The results provided valuable information indicating selection for the BMP4 genotype might increase growth and muscularity in Barki lambs.

Assessment of Genetic Variability of Citrus tristeza virus by SSCP and CE-SSCP.

Single-strand conformation polymorphism (SSCP) is a popular method used to study the genetic heterogeneity and population variability of Citrus tristeza virus (CTV) isolates. It is a simple, low-cost, and highly specific method for mutation detection of specific genes, mostly of the CTV major coat protein gene (p25). The technique is based on a comparison on polyacrylamide gel of electrophoretic profiles of single-stranded (ss) DNA sequences in terms of their spatial conformation. SSCP involves cDNA synthesis and amplification of the target gene, denaturation of single strands, and electrophoresis in non-denaturing conditions. The ssDNAs can be afterward visualized by staining the polyacrylamide gel. Alternatively, using fluorescently labeled primers, the procedure can be performed in automated sequencers equipped with an appropriate capillary (CE-SSCP), which increases the potential of high-throughput analysis, precision, and the reproducibility of results. CE-SSCP can be also directly applied to the virus particles obtained by elution from ELISA plates or tissue-print membranes.

A note on cloning and analysis of the osteopontin gene with egg quality traits in two Chinese domestic ducks.

1. Osteopontin (OPN) is a highly phosphorylated acidic glycoprotein that plays a crucial role in eggshell formation. In this study, an 893-bp cDNA sequence of the OPN gene, which encodes 180 amino acids, was obtained. 2. Polymorphisms of the OPN gene were analysed with DNA sequencing and polymerase chain reaction-single-strand conformation polymorphism methods in two Chinese domestic laying ducks (Jingding n = 100, Youxian n = 478, respectively). 3. One polymorphism was identified in exon 7 (NM_ 004676534.1:c.267T>C) of the OPN gene, with three genotypes: TT (both T allels weren't mutated (wild type)), TC (one T allel was mutated to C (heterozygote genotype)) and CC. (both T allels were mutated to C (homozygote 20 genotype)) Association analysis with egg quality traits in the two Chinese domestic laying ducks showed that the ducks with the CC genotype had significantly greater eggshell strength and eggshell thickness (p < 0.05). Hence, the exon 7 267T>C polymorphism of the OPN gene is a potentially valuable genetic marker for laying duck breeding programmes.

Prion protein testis specific (PRNT) gene polymorphisms and transcript level in ovine spermatozoa: Implications in freezability, fertilization and embryo production.

An essential role of prion protein testis specific (PRNT) and prion protein 2 dublet (PRND) genes in the male reproductive function has been highlighted, although a deeper knowledge for the mechanisms involved is still lacking. Our goal was to determine the importance of the PRNT haplotypic variants and mRNA expression levels in ovine spermatozoa freezability and ability for fertilization and embryo developmental processes. Their association with the PRND gene polymorphisms was also analyzed. DNA from rams belonging to three Portuguese sheep breeds (n = 28) was screened by single-strand conformation polymorphism (SSCP) analysis to identify the PRNT and PRND polymorphisms. Semen collected from these rams was cryopreserved and fertility traits evaluated. The SSCP analyses revealed polymorphisms in the codons 6, 38, 43 and 48 of the PRNT coding region - respectively c.17C > T (p.Ser6Phe, which disrupts a consensus arginine-X-X serine/threonine motif); c.112G > C (p.Gly38 > Arg); and synonymous c.129T > C and c.144A > G. The polymorphisms in codons 6, 38 and 48 occur simultaneously while the one in codon 43 occurs independently. Six haplotypes were identified in the PRNT coding region, resulting in three different amino acid polymorphic variants (6S-38G-43C-48V, S6F-G38R-43C-48V and 6F-38R-43C-48V). The PRNT gene mRNA transcript level in spermatozoa was related to the identified haplotypic variants, either considering the codons 6-38-48 (P ≤ 0.0001) or the codon 43 alone (P ≤ 0.0001) or altogether (P ≤ 0.0001). An interaction between PRNT haplotypes and PRND genotypes on PRNT transcript level was also identified (P = 0.0003). Rams carrying the 17C-112G-144A PRNT haplotype had sperm with the highest post-thawed individual motility (P ≤ 0.03). Combined PRNT and PRND polymorphic variation influenced the post-thawed individual motility (P = 0.01). The male PRNT haplotypic, either considering the codons 6-38-48 and 43 altogether or the codon 43 alone, interfered (P ≤ 0.04) in embryo production rates. In conclusion, our data confirm that the PRNT gene is highly polymorphic in sheep and that the PRNT and PRND genotypes are associated. The identified polymorphisms of PRNT coding region seems to interfere on the ram spermatozoa mRNA transcript level and on male fertility, specifically in sperm freezability and ability for embryo development.

Structural polymorphism of a cytosine-rich DNA sequence forming i-motif structure: Exploring pH based biosensors.

Sequence recognition and conformational polymorphism enable DNA to emerge out as a substantial tool in fabricating the devices within nano-dimensions. These DNA associated nano devices work on the principle of conformational switches, which can be facilitated by many factors like sequence of DNA/RNA strand, change in pH or temperature, enzyme or ligand interactions etc. Thus, controlling these DNA conformational changes to acquire the desired function is significant for evolving DNA hybridization biosensor, used in genetic screening and molecular diagnosis. For exploring this conformational switching ability of cytosine-rich DNA oligonucleotides as a function of pH for their potential usage as biosensors, this study has been designed. A C-rich stretch of DNA sequence (5'-TCCCCCAATTAATTCCCCCA-3'; SG20c) has been investigated using UV-Thermal denaturation, poly-acrylamide gel electrophoresis and CD spectroscopy. The SG20c sequence is shown to adopt various topologies of i-motif structure at low pH. This pH dependent transition of SG20c from unstructured single strand to unimolecular and bimolecular i-motif structures can further be exploited for its utilization as switching on/off pH-based biosensors.

SNPs of CD14 change the mastitis morbidity of Chinese Holstein.

Gram­negative (GN) bacterial infection is a main cause of bovine mastitis. The cluster of differentiation (CD) 14 gene serves an essential role in GN bacterium­induced innate immune response. CD14 works as a bacterial lipopolysaccharide (LPS) receptor, combines with LPS­liposaccharide binding protein complex, and causes cellular activation. However, the effects of CD14 single nucleotide polymorphisms (SNPs) on morbidity of clinical mastitis remain unclear. In the present study, To investigate the polymorphisms of CD14 gene and its effects on cows' susceptibility to mastitis, polymerase chain reaction­single­strand conformation polymorphism (PCR­SSCP) assay was used to detect SNPs of CD14 gene in 134 Chinese Holsteins. SNPs were identified in PCR products amplified with 3 sets of primers in CD14 exon 2. A total of three SNPs were located in that exon: g.528 A→C (147Ser→Arg) in allele B; g.612 A→G (175Asn→Asp) in allele D; and g.1022 A→G in allele F (synonymous mutation). The SNPs in alleles B and D affected the secondary structure of CD14. A 3­dimensional (3D) structural analysis predicted three potential protein forms with a similar structure and indicated that the changes of the above­mentioned alleles were on the concave surface of the protein. In more detail, 147 Ser→Arg induced a protein kinase C phosphorylation site to move forward, as assessed by the motif analysis. The morbidity rate of AB (mixed type g.528 A/C) and CD (mixed type g.612 A/G) was the highest among all genotypes presented in the current study, and via of tumor necrosis factor­α and interleukin­6 mRNA levels were upregulated in animals of this genotype compared with others. Taken together, the CD14 SNPs identified in the present study, may be closely associated with the morbidity of mastitis.

Accurate and effective multidrug-resistant Mycobacterium tuberculosis detection method using gap-filling ligation coupled with high-resolution capillary electrophoresis-based single strand conformation polymorphism.

Tuberculosis (TB) has severely threatened public health via emerging multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) strains. For effective TB treatment, rapid, accurate, and multiplex detection of drug resistance is extremely important. However, conventional methods for TB diagnosis are time consuming and have a limited effect on treatment. Nucleic acid-based molecular detection methods have been developed as an effective MDR/XDR-TB diagnosis technology. Among the nucleic acid-based methods, ligation-dependent methods are attractive as MDR/XDR-MTB detection technologies, but multiplex analysis is limited by the detection method. Although an electrophoresis-based method is considered for multiple target detection because it is free from the errors pertaining to hybridization-based systems, the procedure of multiplex analysis is quite complicated owing to the DNA size-based separation system. In this study, we report an accurate, rapid, and simple multiple MDR/XDR-MTB detection technology using gap-filling ligation reaction coupled with high-resolution capillary electrophoresis-based single-strand conformation polymorphism. Using this system, rapid and accurate MDR/XDR-MTB detection is feasible via similar length probes without the complicated step of probe design. We found that this method could accurately and effectively detect highly polymorphic regions in specific codons associated with drug resistance.

Identification of the cysteine-rich 61 (CYR61) gene variations in osteosarcoma patients.

BACKGROUND/AIM: Osteosarcoma requires an angiogenesis process. CYR61 is one of the extracellular signaling molecules that promote angiogenesis, tumor growth, and the malignancy of osteosarcoma. In the present study, we investigate the CYR61 gene variations in osteosarcoma and their correlations with clinicopathological findings. MATERIALS AND METHODS: We performed variation analysis of the CYR61 gene in 58 patients with osteosarcoma. With an aim to ascertain the variety of variations in exons 2, 3, 4, and 5 of the CYR61 gene in osteosarcoma, we did a PCR-SSCP followed by DNA sequencing. RESULTS: In osteosarcoma the CYR61 gene variations found were 18.96% (11/58) in exon 2, 3.44% (2/58) in exon 3, 8.62% (5/58) in exon 4, and 15.51% (9/58) in exon 5. In our variation analysis, we detected one missense variation in exon 2 (Arg47Trp), one silent variation in exon 3 (Lys152Lys), one missense variation in exon 4 (Phe213Leu), and two missense variations in exon 5 (Gly315Arg and Asp339Asn). The overall CYR61 variation frequency in exons 2, 3, 4, and 5 was determined to be 46.55% (27/58). CONCLUSION: Our study specifies the role of CYR61 gene variation in osteosarcoma. The result signifies that CYR61 might be used as a prognostic/diagnostic marker in osteosarcoma patients.

Evolution of Gene Expression Balance Among Homeologs of Natural Polyploids.

Polyploidy is a major evolutionary process in eukaryotes, yet the expression balance of homeologs in natural polyploids is largely unknown. To study this expression balance, the expression patterns of 2180 structurally well-characterized genes of wheat were studied, of which 813 had the expected three copies and 375 had less than three. Copy numbers of the remaining 992 ranged from 4 to 14, including homeologs, orthologs, and paralogs. Of the genes with three structural copies corresponding to homeologs, 55% expressed from all three, 38% from two, and the remaining 7% expressed from only one of the three copies. Homeologs of 76-87% of the genes showed differential expression patterns in different tissues, thus have evolved different gene expression controls, possibly resulting in novel functions. Homeologs of 55% of the genes showed tissue-specific expression, with the largest percentage (14%) in the anthers and the smallest (7%) in the pistils. The highest number (1.72/3) of homeologs/gene expression was in the roots and the lowest (1.03/3) in the anthers. As the expression of homeologs changed with changes in structural copy number, about 30% of the genes showed dosage dependence. Chromosomal location also impacted expression pattern as a significantly higher proportion of genes in the proximal regions showed expression from all three copies compared to that present in the distal regions.

Highly multiplex and sensitive SNP genotyping method using a three-color fluorescence-labeled ligase detection reaction coupled with conformation-sensitive CE.

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost-effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation-dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error-prone hybridization-based detection, the multiplex assay process is complicated because of the size-based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high-resolution CE-SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar-sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three-color fluorescence-labeled probes.

Intratumoral Heterogeneity of Frameshift Mutations in MECOM Gene is Frequent in Colorectal Cancers with High Microsatellite Instability.

MECOM gene, also known as EVI, encodes a transcriptional regulator involved in hematopoiesis, apoptosis, development and proliferation. In blood system, MECOM is considered an oncogene, but in solid tumors it has both oncogenic and tumor suppressor activities. Low frequent somatic mutations of MECOM have been detected in many cancers including colorectal cancers (CRC), but the mutation status with respect to the microsatellite instability (MSI) has not been studied. There is an A7 mononucleotide repeat in MECOM coding sequences that could be a mutation target in the cancers with MSI. We analyzed the A7 of MECOM in 79 CRCs with high MSI (MSI-H) and 65 microsatellite stable/low MSI (MSS/MSI-L) CRCs by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found MECOM frameshift mutations in 6 (7.6 %) CRCs with MSI-H, but not in MSS/MSI-L cancers (0/65) (p < 0.025). We also analyzed intratumoral heterogeneity (ITH) of the MECOM frameshift mutation in 16 CRCs and found that four CRCs (25.0 %) harbored regional ITH of the frameshift mutations. Our data indicate that MECOM gene harbors both somatic frameshift mutations and mutational ITH, which together may be features of CRC with MSI-H.

Haplotypic variation in the UCP1 gene is associated with milk traits in dairy cows.

Uncoupling protein-1 (UCP1) plays a role in the regulation of body temperature, metabolic rate and energy expenditure in animals. While variation in UCP1 and its phenotypic effect has been investigated in humans and sheep, little is known about this gene in cattle. In this study, four regions of bovine UCP1 were investigated in 612 Holstein-Friesian × Jersey (HF × J) dairy cows using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analyses. In the four regions of the gene analysed, a total of 13 SNPs were detected. Three sequences (a, b and c) were found in Region-2 and three sequences (A, B and C) were found in Region-4, and these were assembled into three (a-B, b-B and c-A) common and three (b-C, c-B and c-C) rare haplotypes. Of the three common haplotypes, b-B and c-A were associated (P < 0·007 and P < 0·043, respectively) with increased milk yield and tended to be associated (P < 0·085 and P < 0·070, respectively) with decreased fat percentage. Cows with genotype b-B/a-B produced more milk (P < 0·004), but with a lower percentage of fat (P < 0·035) and protein (P < 0·038) than cows with genotype a-B/a-B. Cows of genotype a-B/c-A had milk of low fat percentage (P < 0·017), but tended to produce more milk (P < 0·059) than cows of genotype a-B/a-B. This suggests that UCP1 affects milk yield, milk fat percentage and milk protein percentage.

POLYMORPHISM OF THE PROLACTIN GENE AND ITS EFFECT ON FIBER TRAITS IN GOAT.

The prolactin gene (PRL) is a potential candidate gene for the goat cashmere traits in marker-assisted selection. Thus, the aim of this study was to detect PRL gene polymorphism and its association with fiber traits in 200 Raini cashmere goats native to the south-east of Iran. A 196-bp fragment encoding exon 5 within the goat PRL gene was amplified using PCR specific primers. The amplification products were subjected to the single stranded conformation polymorphism (SSCP) analysis. Three different SSCP banding patterns (CC, AC and AA) were observed in exon 5 of the caprine PRL gene. The pattern frequencies for CC, AC and AA were 0.39, 0.38 and 0.23 and frequencies of the A and C alleles were 0.42 and 0.58, respectively. The genotypic distributions did not deviate from the Hardy-Weinberg equilibrium (P> 0.05). The number of observed alleles, number of effective alleles, expected heterozygosity, observed heterozygosity, mean of heterozygosity, expected homozygosity, observed homozygosity, Nei's index and Shanon's index were 2.0, 1.9, 0.48, 0.38, 0.48, 0.51, 0.61, 0.48 and 0.68, respectively. Results of association between genotypes and fiber traits indicated that the CC genotype had the highest fiber length compared with the AA and AC genotypes (P < 0.05) while there was no significant association between the PRL gene genotypes and fiber diameter. These results imply that the PRL gene polymorphism can be used as a molecular marker to improve fiber production without a negative effect on fiber diameter.