Stored alcohol and fatty acid intermediates and the biosynthesis of sex pheromone aldehyde in the moth Chloridea virescens.

In most species of moths, the female produces and releases a volatile sex pheromone from a specific gland to attract a mate. Biosynthesis of the most common type of moth sex pheromone component (Type 1) involves de novo synthesis of hexadecanoate (16:Acyl), followed by modification to various fatty acyl intermediates, then reduction to a primary alcohol, which may be acetylated or oxidized to produce an acetate ester or aldehyde, respectively. Our previous work on the moth Chloridea virescens (Noctuidae) showed that females produce 90% of the major pheromone component, (Z)-11-hexadecenal (Z11-16:Ald), via a direct and rapid route of de novo biosynthesis with highly labile intermediates, and ca. 10% from an indirect route that likely mobilizes a pre-synthesized 16-carbon skeleton, possibly, (Z)-11-hexadecenoate (Z11-16:Acyl) or hexadecanoate (16:Acyl). In this paper, we use stable isotope tracer/tracee techniques to study the dynamics of the precursor alcohol (Z)-11-hexadecenol (Z11-16:OH) and stores of Z11-16:Acyl and 16:Acyl to determine their roles in biosynthesis of Z11-16:Ald. We found: (i) that intracellular Z11-16:OH is synthesized at roughly the same rate as Z11-16:Ald, indicating that translocation and oxidation of this moiety does not rate limit biosynthesis of Z11-16:Ald, (ii) intracellular Z11-16:OH consists of two pools, a highly labile one rapidly translocated out of the cell and converted to Z11-16:Ald, and a less labile one that mostly remains in gland cells, (iii) during pheromone biosynthesis, net stores of Z11-16:Acyl increase, suggesting it is not the source of Z11-16:Ald produced by the indirect route, and (iv) no evidence for the gland synthesizing stored 16:Acyl prior to (up to 2 days before eclosion), or after, synthesis of pheromone commenced, suggesting the bulk of this stored moiety is synthesized elsewhere and transported to the gland prior to gland maturation. Thus, the pheromone gland of C. virescens produces very little stored fat over its functional lifetime, being optimized to produce sex pheromone.

Some Factors Influencing Calling Behavior and Mass Emission Rate of Sex Pheromone from the Gland of the Moth Chloridea virescens.

To attract a mate, females of most moth species synthesize and emit sex pheromone from a specific gland in a behavior termed "calling". In a broad temporal sense, calling behavior and pheromone synthesis are synchronized through the overlap of their circadian rhythms. However, the limited amount of pheromone a female produces each day must be managed so that pheromone is emitted at a sufficient (to attract males) mass emission rate (MER) over the entire calling period, typically many hours. We are studying pheromone synthesis and emission in the moth Chloridea (formerly Heliothis) virescens (family Noctuidae). One way that female C. virescens manage pheromone over their calling period is by calling intermittently; the period between calling bouts allows females to replenish pheromone, and resume calling at high MERs. However, militating against replenishment is loss of pheromone through putative catabolism. In this paper, we examined three aspects pertaining to pheromone MER in C. virescens: (i) the effect of adult feeding on calling behavior, (ii) the effect of certain behavioral/physical parameters on MER, and (iii) the relative loss (putative catabolism) of pheromone in retracted (non-calling) and everted (calling) glands. We found that (i) adult feeding increases calling duration, consistent with the known concomitant increase in pheromone production, (ii) various physical factors relating to the gland, including degree of eversion (surface area), orientation to airstream, and air velocity over the gland influence MER, and (iii) putative catabolism occurs in both retracted and everted glands, but substantially less pheromone is lost in the everted gland primarily because of the high MER when the gland is first everted. Together, these data demonstrate that, over the calling period, the efficient use of pheromone for emission by female C. virescens is dependent on the interaction among synthesis, storage, catabolism, and calling behavior.

Sex pheromone biosynthesis, storage and release in a female moth: making a little go a long way.

Moth pheromone research has pioneered much of our understanding of long-distance chemical communication. Two important characteristics of this communication have, however, remained largely unaddressed: the release of small quantities of pheromone by most moth species, despite potential advantages of releasing greater amounts, and the intermittency of release in some species, limiting the time of mate attraction. We addressed the proximate mechanisms underlying these characteristics by manipulating biosynthesis, storage and release of pheromone in females of the noctuid moth Chloridea virescens. We found that (i) mass release is determined by pheromone mass on the gland surface; (ii) amounts synthesized are limited by pheromone biosynthesis activating neuropeptide concentration, not precursor availability; (iii) some gland structural feature limits mass release rate; (iv) intermittent calling enables release at a mass rate greater than biosynthetic rate; and (v) at typical mass release rates, the periodicity of pheromone availability on the gland surface roughly matches the periodicity (intermittency) of calling. We conclude that mass release in C. virescens and possibly many other species is low because of constraints on biosynthesis, storage and gland structure. Further, it appears the behaviour of intermittent calling in C. virescens may have evolved as a co-adaptation with pheromone availability, allowing females to release pheromone intermittently at higher mass rates than the biosynthesis rate.

The Effect of Pheromone Synthesis and Gland Retraction on Translocation and Dynamics of Pheromone Release in the Moth Chloridea virescens.

Most species of moths use a female-produced sex pheromone to bring mates together. Typically, sex pheromone is synthesized in a specialized gland and released during the behavior of "calling", in which the ovipositor and gland are extruded, allowing pheromone to evaporate. Although there has been much study on how a gland makes specific pheromone components, we know relatively little about how it actually functions with regard to synthesis, storage and release. In this paper, we investigated three aspects of gland function in the noctuid moth Chloridea virescens (Fabricius): (i) whether translocation of pheromone from site of synthesis to release is dependent on calling or ovipositor movement, (ii) whether pheromone synthesis rate limits release and (iii) how intermittent calling (observed in this and other species) might affect the dynamics of release rate. Firstly, by manipulating the gland to simulate calling (extruded) or non-calling (retracted), we showed that pheromone translocation occurred regardless of whether the gland was retracted or extruded. Secondly, by manipulating pheromone production, we found that females that produced more pheromone had higher release rates. It was especially noticeable that females had a higher release rate at the start of calling, which dropped rapidly and leveled off over time. Together, these data suggest that intermittent calling in C. virescens (and other species) may function to allow females to replenish pheromone stores on the gland surface between calling bouts, so that brief, high release rates occur at the start of a calling bout; thus, potentially increasing a female's chances of attracting a mate.

HPV genotype distribution and age correlation in a selected Italian population undergoing conization.

BACKGROUND: Detection and genotyping of human papillomavirus (HPV) has gained increasing importance in cervical cancer prevention and treatment of cervical intraepithelial neoplasia (CIN). This study aims to determine the HPV type distribution in cervical specimens obtained from women diagnosed with CIN. We evaluated in a selected Italian population the distribution of HPV genotypes. METHODS: Cervical samples were collected from women undergoing laser CO2 conization for high grade at Colposcopic Laser Surgery Unit of the Careggi University Hospital and at the Colposcopy Service of Local Health Unit Toscana Centro in Florence, Italy, between September 2014 and February 2017. HPV genotyping was performed using the LINEAR ARRAY® HPV Genotyping Test. RESULTS: Three hundred and six patients were enrolled. HPV infection was detected on 244 samples (79.7%). A different rate of mono- and poly-infections was observed, with higher poly-infection rates in younger women. Moreover, depending on different age groups (clustered in 5-years interval from 22 to 69 years old) significant different distribution of HPV was fund as genotype, phylogenetic type and cancer-related risk. CONCLUSIONS: Our results suggest that some physiological conditions (i.e. menopause), could influence selection and clearance of specific HPV genotypes. The results of this study represent the basis for supporting the HPV genotyping as clinical tool providing benefits in the management of women with high CIN grade.

Advancing Biologics Development Programs with Legacy Cell Lines: Advantages and Limitations of Genetic Testing for Addressing Clonality Concerns Prior to Availability of Late Stage Process and Product Consistency Data.

The bioprocessing industry uses recombinant mammalian cell lines to generate therapeutic biologic drugs. To ensure consistent product quality of the therapeutic proteins, it is imperative to have a controlled production process. Regulatory agencies and the biotechnology industry consider cell line "clonal origin" an important aspect of maintaining process control. Demonstration of clonal origin of the cell substrate, or production cell line, has received considerable attention in the past few years, and the industry has improved methods and devised standards to increase the probability and/or assurance of clonal derivation. However, older production cell lines developed before the implementation of these methods, herein referred to as "legacy cell lines," may not meet current regulatory expectations for demonstration of clonal derivation. In this article, the members of the IQ Consortium Working Group on Clonality present our position that the demonstration of process consistency and product comparability of critical quality attributes throughout the development life cycle should be sufficient to approve a license application without additional genetic analysis to support clonal origin, even for legacy cell lines that may not meet current day clonal derivation standards. With this commentary, we discuss advantages and limitations of genetic testing methods to support clonal derivation of legacy cell lines and wish to promote a mutual understanding with the regulatory authorities regarding their optional use during early drug development, subsequent to Investigational New Drug (IND) application and before demonstration of product and process consistency at Biologics License Applications (BLA) submission.

Calling Behavior and Sex Pheromone Release and Storage in the Moth Chloridea virescens.

Female moths release sex pheromone to attract mates. In most species, sex pheromone is produced in, and released from, a specific gland. In a previous study, we used empirical data and compartmental modeling to account for the major pheromone gland processes of female Chloridea virescens: synthesis, storage, catabolism and release; we found that females released little (20-30%) of their pheromone, with most catabolized. The recent publication of a new pheromone collection method led us to reinvestigate pheromone release and catabolism in C. virescens on the basis that our original study might have underestimated release rate (thereby overestimating catabolism) due to methodology and females not calling (releasing) continuously. Further we wished to compare pheromone storage/catabolism between calling and non-calling females. First, we observed calling intermittency of females. Then, using decapitated females, we used the new collection method, along with compartmental modeling, gland sampling and stable isotope labeling, to determine differences in pheromone release, catabolism and storage between (forced) simulated calling and non-calling females. We found, (i) intact 1 d females call intermittently; (ii) pheromone is released at a higher rate than previously determined, with simulations estimating that continuously calling females release ca. 70% of their pheromone (only 30% catabolized); (iii) extension (calling)/retraction of the ovipositor is a highly effective "on/off' mechanism for release; (iv) both calling and non-calling females store most pheromone on or near the gland surface, but calling females catabolize less pheromone; (v) females are capable of producing and releasing pheromone very rapidly. Thus, not only is the moth pheromone gland efficient, in terms of the proportion of pheromone released Vs. catabolized, but it is highly effective at shutting on/off a high flux of pheromone for release.

Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics.

Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

Production and Distribution of Aldehyde and Alcohol Sex Pheromone Components in the Pheromone Gland of Females of the Moth Chloridea virescens.

Aldehydes are components of many moth sex pheromones, and are thought to be produced from analogous alcohols by oxidase(s) in the cell membrane or the gland cuticle. This implies that the two types of components are produced and/or stored in different parts of the gland: alcohols in cells and aldehydes in cuticle. Few studies have investigated the distribution of components in moth pheromone glands. Using rinse/extract sampling, stable isotope tracer/tracee methods, and decapitation/ pheromone biosynthesis activating neuropeptide stimulation, we studied production and distribution of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-hexadecenol (Z11-16:OH) in the gland of Chloridea virescens (formerly Heliothis virescens). The rinse, which likely sampled the surface and outer cuticle, contained large amounts of aldehyde and small amounts of alcohol. By contrast, the residual extract, which likely sampled cells and less solvent-accessible (inner) cuticle, had large amounts of alcohol and small amounts of aldehyde. When a tracer (U-13C-glucose) was fed to females, the aldehyde had higher isotopic enrichment than the alcohol in the rinse, but not in the residual extract, showing that in the rinse pool, Z11-16:Ald was, on average, synthesized before Z11-16:OH. This is consistent with greater aldehyde than alcohol flux through the cuticle. While our results are consistent with cell/cuticle synthesis sites for alcohol/aldehyde components, we cannot rule out both being synthesized in gland cells. We propose two alternative conceptual models for how site of production, cuticular transport and catabolism/metabolism might explain the relative masses of Z11-16:Ald and Z11-16:OH translocated to the pheromone gland surface in female C. virescens.

The Dynamics of Pheromone Gland Synthesis and Release: a Paradigm Shift for Understanding Sex Pheromone Quantity in Female Moths.

Moths are exemplars of chemical communication, especially with regard to specificity and the minute amounts they use. Yet, little is known about how females manage synthesis and storage of pheromone to maintain release rates attractive to conspecific males and why such small amounts are used. We developed, for the first time, a quantitative model, based on an extensive empirical data set, describing the dynamical relationship among synthesis, storage (titer) and release of pheromone over time in a moth (Heliothis virescens). The model is compartmental, with one major state variable (titer), one time-varying (synthesis), and two constant (catabolism and release) rates. The model was a good fit, suggesting it accounted for the major processes. Overall, we found the relatively small amounts of pheromone stored and released were largely a function of high catabolism rather than a low rate of synthesis. A paradigm shift may be necessary to understand the low amounts released by female moths, away from the small quantities synthesized to the (relatively) large amounts catabolized. Future research on pheromone quantity should focus on structural and physicochemical processes that limit storage and release rate quantities. To our knowledge, this is the first time that pheromone gland function has been modeled for any animal.

Implementation of Plate Imaging for Demonstration of Monoclonality in Biologics Manufacturing Development.

Monoclonality of mammalian cell lines used for production of biologics is a regulatory expectation and one of the attributes assessed as part of a larger process to ensure consistent quality of the biologic. Historically, monoclonality has been demonstrated through statistics generated from limiting dilution cloning or through verified flow cytometry methods. A variety of new technologies are now on the market with the potential to offer more efficient and robust approaches to generating and documenting a clonal cell line.Here we present an industry perspective on approaches for the application of imaging and integration of that information into a regulatory submission to support a monoclonality claim. These approaches represent the views of a consortium of companies within the BioPhorum Development Group and include case studies utilising imaging technology that apply scientifically sound approaches and efforts in demonstrating monoclonality. By highlighting both the utility of these alternative approaches and the advantages they bring over the traditional methods, as well as their adoption by industry leaders, we hope to encourage acceptance of their use within the biologics cell line development space and provide guidance for regulatory submission using these alternative approaches.LAY ABSTRACT: In the manufacture of biologics produced in mammalian cells, one recommendation by regulatory agencies to help ensure product consistency, safety, and efficacy is to produce the material from a monoclonal cell line derived from a single, progenitor cell. The process by which monoclonality is assured can be supplemented with single-well plate images of the progenitor cell. Here we highlight the utility of that imaging technology, describe approaches to verify the validity of those images, and discuss how to analyze that information to support a biologic filing application. This approach serves as an industry perspective to increased regulatory interest within the scope of monoclonality for mammalian cell culture-derived biologics.

Analytical subcloning of a clonal cell line demonstrates cellular heterogeneity that does not impact process consistency or robustness.

During development of a cell line intended to support production of an IgG2 monoclonal antibody, a sequence variant caused by a genetic mutation was identified in the bulk drug substance. Gene copy number analysis together with the level of the observed variant in genomic DNA indicated that the master cell bank was a mixed population of cells; some harboring the variant copy and some mutation free. Since the cell bank had been single-cell cloned, this variant could be used as a biomarker to demonstrate either that the bank was not derived from a single cell, or that the variant was a result of a post-cloning genetic event, leading to a mixed population of cells. The sequence variant was only present in a small percentage of subclones, confirming the hypothesis that the cell bank was indeed a mixed population. Interrogation of subclones via Southern blot analysis revealed that almost all subclones had very similar transgene integrant structures, suggesting that the cell bank was likely derived from a single cell, and the cellular event that yielded the sequence variant was a post-cloning event. Further, there were likely several other post-cloning events that impacted transgene loci, leading to a population of related, yet genetically distinct cells comprising the cell bank. Despite this, the heterogeneous bank performed consistently in a bioprocess across generational age with comparable product quality. These results experimentally demonstrate the heterogeneity of a cell bank derived from a single cell, and its relationship to process consistency. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:602-612, 2018.

Differential Pheromone Sampling of the Gland of Female Heliothis Virescens Moths Reveals Glandular Differences in Composition and Quantity.

By differentially sampling the pheromone gland of females of the moth Heliothis virescens, we explored differences in pheromone on the surface, or outer distal layer(s) of the gland, and that located more proximally. For this, we used two sampling approaches, (i) a solid phase microextraction fiber rub followed by solvent extraction of residual pheromone (SPME rub/extract), and (ii) rapid solvent rinsing followed by solvent extraction of residual pheromone (rinse/extract). The SPME rub showed differences in component ratio between the dorsal and ventral gland surfaces. The rinse sampled a greater amount of pheromone than the SPME rub, sampling the whole gland surface as well as likely deeper into the gland. Compared to the other samplings, pheromone in the rinse was depleted in the minor component; consequently, the corresponding residual extract was highly enriched in the minor component. Further rinses of the gland yielded only small amounts of pheromone, with a similar component ratio as the first rinse, suggesting that the residual pheromone was less accessible and required extraction in solvent to be liberated. Sampling over the photoperiod showed that the more volatile minor component was depleted (relative to the major component) on the surface/outer cuticle over the period when females called. Together, these data suggest that the pheromone is stored, at least in part, on and in the gland cuticle and that distinct pools may be transported to different topographic regions. Females fed with a stable isotope tracer, incorporated label into pheromone in the gland very rapidly, with the labeled pheromone appearing on the gland surface ca. 1 min later.

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process.

Sex pheromone in the moth Heliothis virescens is produced as a mixture of two pools: de novo and via precursor storage in glycerolipids.

Most species of moths use a female-produced volatile sex pheromone, typically produced via de novo fatty acid synthesis in a specialized gland, for communication among mates. While de novo biosynthesis of pheromone (DNP) is rapid, suggesting transient precursor acids, substantial amounts of pheromone precursor (and other) acids are stored, predominantly in triacylglycerols in the pheromone gland. Whether these stored acids are converted to pheromone later or not has been the subject of some debate. Using a tracer/tracee approach, in which we fed female Heliothis virescens U-13C-glucose, we were able to distinguish two pools of pheromone, in which precursors were temporally separated (after and before feeding on labeled glucose): DNP synthesized from a mixed tracer/tracee acetyl CoA pool after feeding, and pheromone made from precursor acids primarily synthesized before feeding, which we call recycled precursor fat pheromone (RPP). DNP titer varied from high (during scotophase) to low (photophase) and with presence/absence of pheromone biosynthesis activating neuropeptide (PBAN), in accord with native pheromone titer previously observed. By contrast, RPP was constant throughout the photoperiod and did not change with PBAN presence/absence. The amount of RPP (6.3-10.3 ng/female) was typically much lower than that of DNP, especially during the scotophase (peak DNP, 105 ng/female). We propose an integral role for stored fats in pheromone biosynthesis, in which they are hydrolyzed and re-esterified throughout the photoperiod, with a small proportion of liberated precursor acyl CoAs being converted to pheromone. During the sexually active period, release of PBAN results in increased flux of glucose (from trehalose) and hydrolyzed acids entering the mitochondria, producing acetyl CoA precursor for de novo fat and pheromone biosynthesis.

Utilization of sequence variants as biomarkers to analyze population dynamics in cloned cell lines.

The inherent nature of cloned CHO cell lines includes the presence of genetic and phenotypic drift that leads to heterogeneous populations. The genetic heterogeneity exhibited by these cells can be exploited to understand the population dynamics of cloned cell lines. Understanding the interplay between heterogeneity, cell culture conditions, and population dynamics will allow for critical assessment of overarching cell line development methods and strategies in terms of population and monoclonality. Sequence variants (SVs) are protein isoforms of the gene-of-interest that contain unintended amino acid substitutions, extensions, or truncations that may contribute to heterogeneity. In this case, SVs are unique sequences in the genome of the integrated transgene that can be used as biomarkers to understand the heterogeneity of a monoclonal cell line and how production process conditions can impact population dynamics. In this study, orthogonal genetic and analytical methods were used to examine the variability of SV levels in four different SV-containing cell lines under varied culture conditions and generational ages. Culture conditions tested had little to no impact on SV levels. However, generational age studies showed two distinct trends: stability of SV levels out to approximately 100 generations in cell lines with higher level SVs (>10%) and a progressive decrease of SV levels as the cells age to approximately 100 generations in cell lines with lower level SVs (<10%). The results suggest that the four SV-containing cell lines fall into two distinct population models; SVs present in the whole population of cells and SVs present in only a sub-population of cells. The data presented here are one of the first studies to not only analyze and compare SV levels in both genetic and protein material but also to utilize SVs as biomarkers to probe distinct populations of cloned cell lines. Biotechnol. Bioeng. 2017;114: 1744-1752. © 2017 Wiley Periodicals, Inc.

Industry view on the relative importance of "clonality" of biopharmaceutical-producing cell lines.

Recently, several health authorities have requested substantial detail from sponsor firms regarding the practices employed to generate the production cell line for recombinant DNA-(rDNA) derived biopharmaceuticals. Two possible inferences from these regulatory agency questions are that (1) assurance of "clonality" of the production cell line is of major importance to assessing the safety and efficacy of the product and (2), without adequate proof of "clonality", additional studies of the cell line and product are often required to further ensure the product's purity and homogeneity. Here we address the topic of "clonality" in the broader context of product quality assurance by current technologies and practices, as well as discuss some of the relevant science and historical perspective. We agree that the clonal derivation of a production cell line is one factor with potential impact, but it is only one of many factors. Further, we believe that regulatory emphasis should be primarily placed on ensuring product quality of the material actually administered to patients, and on ensuring process consistency and implementing appropriate control strategies through the life cycle of the products.

Sex pheromones in mate assessment: analysis of nutrient cost of sex pheromone production by females of the moth Heliothis virescens.

It has been postulated that sex pheromones, in addition to their role in mate recognition and/or finding, may also serve a role in assessment of mate quality. For this, a sex pheromone must give honest information about a signaler's quality, with honesty ensured by a direct metabolic or indirect fitness cost to the signaler. Using a stable isotope tracer-tracee method, we characterized the nutrient pools that fuel sex pheromone production in females of the moth Heliothis virescens, as well as the relative importance of larval- and adult-acquired nutrients to this process. Females used three pools for de novo biosynthesis of sex pheromone, hemolymph trehalose, glycogen (via trehalose) and fat, and produced ca. 25% of pheromone directly from stored (previously synthesized) precursor fatty acids. Pheromone was produced roughly equally from carbohydrate and fat. Adult feeding was very important for pheromone biosynthesis, with a maximum of 65% of de novo biosynthesized pheromone produced from a single adult feed (carbohydrate). Although these nutrient pools are shared with other reproductive physiologies, notably oocyte production, it is unlikely that pheromone production imposes a significant metabolic cost on females, because (i) the amount of nutrients used for pheromone production is negligible compared with that available, (ii) the hemolymph trehalose pool is readily replaceable throughout the adult life, and (iii) in mated females, carbohydrate shortages result in reduced allocation to pheromone.

Increased allocation of adult-acquired carbohydrate to egg production results in its decreased allocation to sex pheromone production in mated females of the moth Heliothis virescens.

Females of most species of moths produce a volatile sex pheromone that attracts conspecific males over distance. In females of the polyandrous moth Heliothis virescens, feeding on carbohydrate (e.g. nectar) supplies precursor, via hemolymph trehalose, for both sex pheromone and egg production. With limited carbohydrate acquisition these two reproductive physiologies might compete for hemolymph trehalose, resulting in an allocation deficit to either sex pheromone or egg production. Using virgin and mated females, which have low and high egg maturation rates, respectively, we fed females a limited diet of (13)C-labeled glucose daily and, using mass isotopomer distribution analysis, determined allocations of adult-acquired carbohydrate (AAC) to newly synthesized pheromone and ovarian and egg fats, our proxies for allocation to egg production. With increased number of feeds, AAC enrichment of hemolymph trehalose increased, as expected. This led to mated females increasing their proportional allocation of AAC to ovarian and egg fats, but decreasing their proportional allocation of AAC to pheromone production. By contrast, virgins increased their proportional allocation of AAC to pheromone production with increased feeds, consistent with increasing AAC enrichment of hemolymph trehalose. These results show that with limited AAC intake, enhanced egg maturation in mated females results in reduced AAC allocation to pheromone production; this does not occur in virgins because of their lower egg maturation rate. This physiological competition for AAC corresponded with decreased pheromone production in mated moths to levels unlikely to attract mates. Therefore, the availability and/or allocation of AAC may be a proximate mechanism underlying the incidence of polyandry in this and other species of moths.

Synthetic rates of key stored fatty acids in the biosynthesis of sex pheromone in the moth Heliothis virescens.

Using a tracer-tracee approach, we fed 1-d-old virgin Heliothis virescens U-(13)C-glucose and analyzed the key labeled fatty acids, (Z)-11-hexadecenoate, hexadecanoate and octadecanoate, known to be intermediates in pheromone biosynthesis, by mass isotopomer distribution analysis. This method allowed determination of enrichment, and fractional (FSR) and absolute (ASR) synthetic rates. As expected, FSRs and ASRs for all three moieties were greater in the scotophase than photophase. However, in whole gland extracts, FSRs and ASRs of (Z)-11-hexadecenoate and hexadecanoate were much lower than those of the major pheromone component, (Z)-11-hexadecenal, determined previously. Since pheromone is made via these acids, we postulated that pheromone was produced directly and very rapidly via a small pool of acyl CoA thioesters of these acids and that the pool of acids we analyzed in our whole gland extract was largely a 'dead end' pool of excess acids (i.e., not converted directly to pheromone) stored in glycerolipids. We tested this by fractionating the whole glandular extract and analyzing the glycerolipid fraction. FSRs and ASRs for the two acids in the glycerolipid fraction were similar to those for the whole gland extract, confirming our postulate. Thus, most acetate produced in the pheromone gland is converted rapidly and directly to pheromone, while excess fatty acids are stored in glycerolipids and remain relatively inaccessible for pheromone production, at least over the two periods studied. Precursor enrichment of octadecanoate was substantially lower than that determined for the two 16-carbon acids and pheromone component. This suggests that hexadecanoate is the principal product of the multi-enzyme complex fatty acid synthase in the gland, and that octadecanoate is formed by subsequent chain elongation of hexadecanoate.