Postal questionnaires are an economical and simple method of data collection for research purposes but are subject to non-response bias. Several studies have explored the effect of monetary and non-monetary incentives on response. Recent meta-analyses conclude that financial incentives are an effective way of increasing response rates. However, large surveys rarely have the resources to reward individual participants. Three previous papers report on the effectiveness of lottery incentives with contradictory results. This study aimed to determine the effect of including a lottery-style incentive on response rates to a postal health survey.Randomised controlled trial. Setting: North and West Birmingham. 8,645 patients aged 18 or over randomly selected from registers of eight general practices (family physician practices). Intervention: Inclusion of a flyer and letter with a health questionnaire informing patients that returned questionnaires would be entered into a lottery-style draw for £100 of gift vouchers. Control: Health questionnaire accompanied only by standard letter of explanation. Main outcome measures: Response rate and completion rate to questionnaire.5,209 individuals responded with identical rates in both groups (62.1%). Practice, patient age, sex and Townsend score (a postcode based deprivation measure) were identified as predictive of response, with higher response related to older age, being female and living in an area with a lower Townsend score (less deprived).This RCT, using a large community based sample, found that the offer of entry into a lottery style draw for £100 of High Street vouchers has no effect on response rates to a postal health questionnaire. Self-completed postal questionnaires are an economical and simple method of data collection for both research studies and audit activity. They are cheaper than telephone or personal interviews  and may Strategies for increasing response rates can be summarised as falling into five broad categories; the covering letter , incentives (cash or other reward), contact (pre-notification and follow-up), mailing  and questionnaire  . Pre-notMuch of the research relating to the effectiveness of specific interventions on response rates in general population surveys was conducted before 1990 and from a market or sociological perspective. More recent work has been conducted on the effects of incentives on increasing response rates in populations of medical professionals. A lottery style incentive (prize of a weekend break) was reported to generate a small increase in response rates from General Practitioners  in Quebec  and cashThe Cochrane Review  identifiThree of the five ,16,18,19Determining the community prevalence of disease is important for needs assessment, service planning and determining the potential economic implications of new treatments. Studies aiming to precisely estimate the prevalence of disease usually require large samples and often use postal questionnaires. A large community mailing to determine the prevalence of Irritable Bowel Syndrome (IBS)  providedThis study was undertaken in Birmingham, a large city in the West Midlands region of the United Kingdom. Ethical approval was obtained from North Birmingham and West Birmingham Research Ethics Committees prior to commencement of the study.Eight general practices in the North and West Birmingham areas were recruited to participate in a health survey designed to determine the prevalence of IBS in the community . All praEstimates were based on the prevalence study within which this trial was embedded and indicated that 8,000 patients should be mailed. This provided 4,000 in each arm of the RCT, sufficient to demonstrate a 4% difference in response rates with 90% power at the 5% significance level, assuming a 60% response rate.To minimise contamination  only one person per domestic address was selected. Practice registers were utilised to generate randomly ordered lists of addresses and then one individual aged 18 or over was randomly selected from each address until the required number of patients had been identified. The prevalence survey, within which this trial was embedded, aimed to recruit a stratified random sample, comprising 1,150 patients from each participating practice to ensure sufficient cases were included from each of the Townsend quartiles. Where the practice was unable to generate 1,150 patients  the maximum number of patients available was included. Randomisation was performed on a 50:50 basis within each practice, to control for practice effects, using a computerised simple random number sequence.All patients selected received a health questionnaire. Questionnaires had three sections; section one requested personal and demographic details in addition to details of personal and family medical history; section two was the SF12 , a validThe intervention group received an identical questionnaire to the control group. The covering letter was also identical apart from the addition of a paragraph explaining that all returned questionnaires would be entered into a draw for a prize of £100 of "High Street shopping vouchers". This letter stressed that entry into the draw was dependent on return rather than completion of a questionnaire, as this was deemed to be more ethically responsible. In addition to the letter, a flyer printed on brightly coloured paper (yellow) was included for intervention patients, highlighting the fact that returned questionnaires would be entered into a draw.All non-responders were re-mailed after 4 weeks. Again, the intervention group received the additional paragraph in the covering letter and an additional flyer. All follow-up mailings included a copy of the questionnaire and a reply paid return envelope. Data handlers were not blinded to the intervention status of responders, but this was not considered to be a source of bias as response rates were the primary outcome.Mailings took place in the period January to July 2001. Mailings were conducted by practice and within each practice, control and intervention patients were mailed on the same day.The principal outcome was the overall response rate. Response rates to initial and follow-up mailings, and numbers of blank responses were also compared between groups.Analysis was on an intention to treat basis. Response rates were compared between the two arms of the trial using chi-squared tests. Predictors of response were identified by logistic regression using backward elimination. Variables entered into the starting model included randomisation arm, age, sex, Townsend score derived from patient postcodes, practice and all two-way interactions with the randomisation arm. Two recent systematic reviews of strategies to influence response rate were identified ,4 and frEight thousand six hundred and forty five patients were included in the trial; 4,325 were randomised to the lottery arm and 4,320 to the control group and a 50:50 split was maintained within each practice.The trial profile is shown in Figure 2 = 0.5, p = 0.48). A further 1197 replied to the reminder mailing; 616 in the intervention arm and 581 in the control arm giving an overall response of 2612/4204 (62.1%) in the intervention arm and 2598/4181 (62.1%) in the control arm . The numbers of questionnaires returned blank was similar for both groups, 197 (4.7%) in the intervention and 217 (5.2%) in the control arm . No objections to the use of an incentive or requests for exclusion from the prize draw were made to the research team or participating general practices.Four thousand and twelve individuals responded to the initial mailing; 1996 (47.5%) in the intervention arm and 2017 (48.2%) in the control group  and provision of the pre-specified prize , and there was therefore a net disbenefit to the use of the incentive.Two previously published relevant studies were identified ,19. ResuThis RCT using a large community sample suggests that using a lottery draw style incentive for £100 of High Street vouchers has no effect on overall response rates to a postal questionnaire asking about health and medical history. Whilst it is possible that the locality of the study  is not typical, the range of practices and Townsend scores of the sample, suggest this is unlikely. It is also possible that a larger incentive or a cash prize, rather than vouchers, may influence response rates. However the value of the incentive offered was comparable to, or greater than, previous studies. The only published study which has shown a significant benefit of a prize draw was of professionals  and we, There was no difference between the groups in the numbers of questionnaires returned blank indicating that the incentive did not just encourage people to return incomplete questionnaires. There was also no difference in the numbers replying to the first mailing. Had the incentive increased numbers replying to the initial mailing, even if it failed to increase response overall, it may have proved cost-effective in terms of reducing the costs of follow-up mailings. Our results indicate that previous research suggesting increased response to initial mailings  is not gThe questionnaire used in this RCT was designed to determine disease prevalence. It is possible that such mailings over-inflate prevalence estimates, as individuals responding may be encouraged to do so because of their vested interest as an individual experiencing disease. Inclusion of an incentive not related to disease status may encourage response from a wider range of the population and may provide more accurate estimates of prevalence. However, this study does not suggest that future health surveys would gain any response benefit from the inclusion of a prize draw incentive. Indeed the addition of the data from this study to that of the two previously published studies based in a health environment ,19 usingA lottery draw style incentive for £100 of High Street vouchers does not affect response rates to a postal health survey, when used in a general population sample. On the basis of this large RCT we would not recommend utilising similar incentives in general population health research.The author(s) declare that they have no competing interests.SW and LR conceived the idea and designed the study. PB contributed to the study design and collected and validated the data. AR completed the analyses. LR wrote the first draft of the manuscript, all other authors made significant contributions to the writing of the manuscript and approved the final version. SW is the guarantor.The pre-publication history for this paper can be accessed here:


Trace heavy metals are essential for a number of metabolic reactions in living systems, but cells walk a fine line between feast or famine. While iron, zinc, cobalt, and manganese, for example, contribute to the catabolic activity of enzymes involved in essential pathways from gene regulation to cell signaling, even a mild surplus of these metals can kill cells and cause a variety of diseases. Maintaining the proper concentration, or homeostasis, of cellular metals requires strict policing of what passes through cell membranes and organelles.One way cells regulate entry is through the hydrophobic lipid (fatty) layer that makes up the cell membrane. While the lipid membrane allows most small fat-soluble or uncharged molecules to simply diffuse through it, nearly all water-soluble molecules, including metal compounds—which typically break down into ions (molecules with positive or negative charge) in solution—rely on either transport or channel proteins to get through.Two types of proteins manage the transport and uptake of iron ions in mammalian cells: the transferrin receptor helps to concentrate iron in discrete intracellular compartments called endosomes, while a protein called divalent metal transporter-1 (DMT1) releases iron into the cytoplasm, where it supports essential metabolic processes. DMT1 also serves to bring dietary iron directly into the intestinal cells involved in iron absorption. DMT1 preferentially carries iron, zinc, copper, and manganese, but not calcium. This selectivity helps strike the right balance of the concentration of these metals in the cell. Recent structural analyses of transporters, however, have raised the possibility that this selectivity may not be as fixed as once thought. Lending support to the notion that the distinctions between transporters and ion channels are blurring, David Clapham, Nancy Andrews, and colleagues report that a mutation causing a single amino acid substitution in the DMT1 metal ion transporter opens a passageway that converts the transporter into a calcium channel.DMT1 is essential for maintaining iron homeostasis and the only molecule known to facilitate transmembrane iron uptake in higher eukaryotes, including humans. It is expressed mainly in epithelial cells of the small intestine, where iron metabolism is monitored, and in endosomes, which release transferrin-imported iron. The Clapham and Andrews groups focused on a mutation in the DMT1 transporter called G185R—which substitutes the arginine (R) amino acid for glycine (G) at a particular location in the protein's amino acid chain, position 185—because the identical mutation has occurred spontaneously in three separate laboratory strains of rodents (two mouse and one rat strain). That a single substitution has arisen independently and persisted in multiple rodent generations suggests it may confer some type of selective advantage.To investigate this idea, the researchers compared the properties of “wild-type” (nonmutant) DMT1 and mutant G185R in laboratory cell lines. They found that cells expressing G185R mutant proteins had much lower levels of iron uptake than cells expressing the nonmutant proteins, but that they also permitted the influx of calcium ions. To see whether the G185R-mediated calcium permeability had a physiological effect on the mice with this mutation, the researchers compared the properties of intestinal epithelial cells taken from the mutant and nonmutant animals. The intestinal cells in the mutant mice showed high levels of the G185R protein and a large current of charged molecules—much as would occur in an ion channel. This current was observed in both the cell lines expressing G185R and the cells extracted from the G185R mutant mice.The G185R mutation, the researchers conclude, appears to either expose or enhance a calcium “permeation pathway” that exhibits the properties of a calcium channel. This transformation appears to offer a selective advantage, since mice engineered without the DMT1 protein die within a week of birth while mice born with the G185R DMT1 mutation can live for over a year. Though the G185R mice exhibit severe iron deficiency, the modest function retained by G185R in combination with the increased influx of calcium may be enough to extend their lifespan. The increased levels of calcium, the researchers propose, may support iron uptake through some other pathway, an advantage that might explain why such a mutation would persist.Whatever mechanism accounts for this advantage, the G185R mutation transforms DMT1 transporter into an “unambiguous” calcium ion channel. Investigating the structural and biochemical properties of this molecular changeling will provide valuable insights into the emerging model of a transporter–channel continuum—which suggests a remarkable adaptability to shifting environmental conditions.


Caenorhabditis elegans and C. briggsae, two Caenorhabditis species with similar reproductive systems and sequenced genomes. We demonstrate that C. briggsae has orthologs of all known C. elegans sex determination genes with one exception: fog-2. Hermaphroditic nematodes are essentially females that produce sperm early in life, which they use for self fertilization. In C. elegans, this brief period of spermatogenesis requires FOG-2 and the RNA-binding protein GLD-1, which together repress translation of the tra-2 mRNA. FOG-2 is part of a large C. elegans FOG-2-related protein family defined by the presence of an F-box and Duf38/FOG-2 homogy domain. A fog-2-related gene family is also present in C. briggsae, however, the branch containing fog-2 appears to have arisen relatively recently in C. elegans, post-speciation. The C-terminus of FOG-2 is rapidly evolving, is required for GLD-1 interaction, and is likely critical for the role of FOG-2 in sex determination. In addition, C. briggsae gld-1 appears to play the opposite role in sex determination  while maintaining conserved roles in meiotic progression during oogenesis. Our data indicate that the regulation of the hermaphrodite germline sex determination pathway at the level of FOG-2/GLD-1/tra-2 mRNA is fundamentally different between C. elegans and C. briggsae, providing functional evidence in support of the independent evolution of self-fertile hermaphroditism. We speculate on the convergent evolution of hermaphroditism in Caenorhabditis based on the plasticity of the C. elegans germline sex determination cascade, in which multiple mutant paths yield self fertility.Somatic and germline sex determination pathways have diverged significantly in animals, making comparisons between taxa difficult. To overcome this difficulty, we compared the genes in the germline sex determination pathways of  C. elegans and C. briggsae provides evidence in support of the convergent evolution of self-fertile hermaphroditism in the Caenorhabditis cladeA comparison of sex determination genes in  Caenorhabditis elegans and Drosophila melanogaster has revealed little about the evolution of the complex pathways that mediate the sexual fate decision in the soma and germline [Sex determination is an ancient and universal feature in metazoans. In spite of this, comparison of distantly related species such as germline ,2,3. ThiC. elegans and C. briggsae represents an ideal case for this type of study, as the sex determination pathway has been well studied in C. elegans and an abundance of sequence information is available for both species [One approach is to perform comparative analysis of sex determination genes in species separated by sufficient evolutionary time to allow for changes in pathway components yet retain comparable somatic and germline morphology and function. The clade containing  species ,5.C. elegans and C. briggsae, while sharing very similar germline and somatic morphology, are separated by approximately 100 million years and are members of a clade that employs multiple mating systems [C. elegans and C. briggsae are self-fertile hermaphrodites that maintain males at a low frequency (androdioecious), whereas the morphologically similar C. remanei and C. sp. CB5161 are obligate female/male (gonochoristic) species [Caenorhabditis species suggests that self-fertile hermaphroditism has evolved independently in C. elegans and C. briggsae from an ancestral male/female state [ systems ,7,8,9,10le state . ImportaC. elegans and C. briggsae, like many other animals, have two sexes specified by the ratio of X chromosomes to sets of autosomes [utosomes ,12,13. Iutosomes ,15. In butosomes ,16,17.C. elegans is the relative activity of two key regulators: tra-2, which promotes the female fate (oocyte), and fem-3, which promotes the male fate (sperm) [tra-2 and fem-3 must be regulated in both males and hermaphrodites to allow spermatogenesis to occur, however the mechanisms by which this regulation occurs differs between the two sexes. In males, her-1 represses tra-2 feminizing activity and raises the relative level of fem-3 activity so that spermatogenesis is continuous [her-1 have no effect on hermaphrodites and her-1 is not expressed in XX animals, a different mechanism is used to allow for the transient production of sperm [A major determinant of germline sexual fate in  (sperm) ,19  and fog-2 (“feminization of germline”)[tra-2 feminizing activity raises the relative level of fem-3 masculinizing activity to promote spermatogenesis ,25  present on the 3′ untranslated region (UTR) of the tra-2 mRNA [tra-2 TGEs results in a loss of GLD-1-mediated translational control and feminization of the germline, such that only oocytes are produced [ogenesis ,29 and eogenesis . GLD-1 iogenesis , includia-2 mRNA ,32  or FOG- gene family and that a fog-2 gene is not present in C. briggsae. Furthermore, double-stranded-RNA-mediated interference (RNAi) of the gld-1 ortholog in C. briggsae results in masculinization of the germline instead of the feminization of the germline phenotype observed in C. elegans. The lack of a potential C. briggsae fog-2 combined with the opposite sex determination function of GLD-1 in C. briggsae indicate that the control of hermaphrodite spermatogenesis, while using most of the same gene products, is fundamentally different between the species and is likely to have evolved independently.We find that 30 of 31 C. elegans and C. briggsae we used reciprocal best BLAST [C. briggsae orthologs of 31 known C. elegans sex determination genes, some of which have been previously identified. The 31 genes included 16 that function only in germline sex determination, seven that function in both somatic and germline sex determination, two that function only in somatic sex determination, and six that coordinate sex determination and dosage compensation. We found that 30 of 31 genes have C. elegans–to–C. briggsae reciprocal best BLAST hits and alignments consistent with a high level of conservation . Importmination ,41  of the C. briggsae genome, or potentially a C. elegans–specific adaptation not present in C. briggsae.A single FOG-2 ortholog could not be resolved by reciprocal best BLAST or by using the reciprocal smallest distance algorithm , which uC. elegans with more than 100 members referred to as FTR proteins [C. briggsae, making the identification of a single functionally equivalent ortholog from a large number of paralogs difficult. Therefore, to discern the relationships among C. elegans and C. briggsae FTR family members, 30 C. elegans and C. briggsae FTR proteins or protein predictions closely related to FOG-2 were used to generate a neighbor-joining phylogeny. The remaining, more diverged FTR members from either species were not included in the phylogeny to avoid long branch attraction [FOG-2 is part of a large, highly diverged F-box- and DUF38/FTH-containing protein family in proteins ,36. The traction .C. elegans and C. briggsae FTR phylogeny reveals that all of the C. elegans FOG-2 relatives form a single clade and all of the C. briggsae relatives a distinct clade. An unrooted radial phylogram illustrating C. elegans and C. briggsae FTR relationships is presented in C. elegans FOG-2 were present in C. briggsae the expectation is that it would have clustered with the C. elegans proteins. Contrary to this, the phylogenetic separation of C. elegans and C. briggsae FTR family members into distinct lineages indicates that extensive expansion in the FTR family occurred post-speciation and that C. elegans and C. briggsae FTR genes do not have one-to-one orthologous relationships.The C. briggsae fog-2 homolog were present in the less than 2% of the genome sequence that is not present in the final assembly or if the fog-2 ortholog diverged sufficiently such that the computational methods were not able to distinguish between orthologous and paralogous relationships. To address these possibilities we used low-stringency cross-species Southern blotting in an effort to identify closely related fog-2-like sequences in unsequenced portions of the C. briggsae genome, and we used conserved synteny in an attempt to identify a diverged fog-2 ortholog that might reside in the same genomic location. Both approaches were used to effectively identify other diverged sex determination genes from C. briggsae  prior to the release of the C. briggsae genome sequence [The above results could be misleading if a closely related sequence ,43,44.C. elegans fog-2 probe and a fem-2 positive control probe against C. briggsae genomic DNA. Under conditions that detected cross-species hybridization with the C. elegans fem-2 probe against C. briggsae genomic DNA [C. briggsae signal was observed with the C. elegans fog-2 probe  were used to query C. briggsae contigs. The genes srg-34, sec-23, and psa-1 are highly conserved across metazoans and have reciprocal best BLAST hits in C. briggsae  have occurred between fog-2 and ftr-1 relative to other C. elegans/C. briggsae best BLAST orthologs. Consistent with a recent duplication, the Ks for fog-2/ftr-1 is not saturated (Ks = 0.36) whereas the average Ks for reciprocal best BLAST hits between C. elegans and C. briggse is saturated (Ks = 1.72) [The closest relative of briggsae   .fog-2 and ftr-1 arose from a relatively recent local duplication within C. elegans strongly supports the contention that fog-2 is not present in C. briggsae. These results imply that C. briggsae must regulate hermaphrodite spermatogenesis differently than C. elegans.The finding that tra-2 3′ UTR translational repression complex. The RNA-binding protein GLD-1 makes direct contact with the tra-2 3′ UTR, and FOG-2 is recruited to the complex via its interaction with GLD-1 [fog-2 and ftr-1  at nucleotide 805 shifts the reading frame of FOG-2 relative to FTR-1 and results in a region of low similarity between the proteins  A–4C. Bef A–4C. Beidentity B and 4C.proteins B. A secoproteins C.The dramatic differences between the FOG-2 and FTR-1 C-terminal regions suggested a connection between the unique functionality of FOG-2 in sex determination and the highly diverged C-terminal region. Since FOG-2 interacts with GLD-1 and both are required for the promotion of the male germ cell fate in the hermaphrodite, we determined whether the diverged FOG-2 C-terminal region was necessary for its interaction with GLD-1 . Progresftr-1 with exon 4 from fog-2 allowed the chimera to interact with GLD-1  to alterations that do not change the amino acid (Ks) [a/Ks ratios that are equal to or less than one are indicative of neutral or purifying selection, where substitutions that change amino acids offer no fitness advantage or result in lowered fitness. In contrast, Ka/Ks ratios greater than one, common in rapidly evolving genes, are indicative of positive selection, where non-synonymous changes offer some fitness advantage and are fixed at a higher rate than synonymous substitutions [Gene duplication provides the raw material for the evolution of novel adaptations, having been implicated in the diversity of the host–pathogen immune response, rapid onset of insecticide resistance, and diversity of vertebrate body plans . Rapidlycid (Ks) ,50. Ka/Kitutions .fog-2/ftr-1 duplication we compared Ka/Ks ratios between fog-2, ftr-1, and the five FTR genes closest to fog-2 in C. elegans. Pairwise comparisons of codon-delimited full-length coding sequences closely related to fog-2 suggest that purifying selection dominates along the fog-2 branch, as all comparisons produced Ka/Ks ratios less than one (mean = 0.46). However, while the overall Ka/Ks ratio for fog-2/ftr-1 is not indicative of positive selection (mean = 0.58), sliding-window Ka/Ks ratio estimates [fog-2 and ftr-1 indicate that the highly diverged C-terminal region of FOG-2/FTR-1 contains residues under positive selection  (a/Ks (PAML and codeml [To determine the selection acting on the stimates  for fog-s 33–37) . An alted codeml ) confirmfog-2 [C. elegans FTR genes in functioning with GLD-1 in germline sex determination. Given the specificity of the FOG-2/GLD-1 interaction in C. elegans, phylogenetic analysis of FTR proteins  had gonad morphology identical to wild-type  and fem-3 (masculinizing) apparently play the same somatic roles in both species, and their epistatic relationship appears to be conserved [RNAi and transgenic experiments have suggested that while mination ,45. Theronserved .fog-2 in C. briggsae is intriguing. fog-2 arose as a consequence of recent C. elegans–specific gene duplication events, and none of the closely related C. elegans fog-2 paralogs can compensate for loss of fog-2 in sex determination [C. briggsae FTRs are involved in GLD-1/tra-2-mRNA-mediated promotion of hermaphrodite spermatogenesis. Since fog-2 is essential for the promotion of spermatogenesis in C. elegans hermaphrodites and is not present in C. briggsae, the direct implication is that specification of the male germ cell fate in C. briggsae hermaphrodites is fundamentally different from that in C. elegans and that it evolved independently.Within the context of general conservation of sex determination pathway components and conserved key epistatic relationships, the absence of mination . Thus, iC. elegans lineage, it is unclear whether fog-2 retains an ancestral function in sex determination and ftr-1 has changed/drifted away or, alternatively, whether ftr-1 represents the ancestral function and fog-2 has recently evolved a role in sex determination  it is unlikely that changes in GLD-1 are responsible for a new mRNA target in C. briggsae. Instead, it is more likely that one or more new target mRNAs have acquired sequences that direct GLD-1 binding and translational repression. The requirements for GLD-1 binding are only just being elucidated, with a hexanucleotide sequence being one important feature amid otherwise diverse GLD-1 binding regions [Another possibility is that  regions ,55. ThusCaenorhabditis and other lineages of Rhabditid nematodes from an ancestral female/male state [tra-2 mRNA is fundamentally different between C. elegans and C. briggsae. This raises the question, how might the transition from the ancestral female/male to hermaphrodite/male system of reproduction have occurred multiple times within the Caenorhabditis clade?Current phylogenetic data suggest that hermaphroditism evolved independently in le state ,10,11,60C. elegans allow both sperm that is introduced by mating and sperm that develops within the female gonad of the hermaphrodite to be effectively used in reproduction [C. briggsae and C. remanei  to induce oocyte maturation and ovulation [Caenorhabditis indicates that major changes in anatomy and reproductive physiology are not necessary in the transition from female/male to hermaphrodite/male reproduction.The anatomy and reproductive physiology of oduction ,61,62. Eoduction . Not onlvulation ,64. ThisC. elegans has suggested that transitions between mating systems may not be difficult and that the overall sex determination pathway reflects selection for a particular mating system rather than a constant regulatory mechanism [C. elegans from hermaphrodite/male to female/male reproduction  in late larvae/adults, self fertility would be possible. After the establishment of self fertility, there would likely be strong selection for additional genetic events that would optimize self-fertile brood size [C. elegans and C. briggsae germline sex determination pathways.Given the conservation of anatomy and reproductive physiology, an initial conversion from an ancestral ood size  and resuC. elegans, the relative levels of TRA-2 feminizing to FEM-3 masculinizing activity appear to be the major regulatory point for the sperm-then-oocyte pattern. There is no a priori reason for TRA-2 or FEM-3 to be the major focus of regulation to achieve hermaphroditism in C. briggsae; if one of these is the focus, then at least some of the regulation must differ between C. elegans and C. briggsae, given the absence of fog-2 and the changed role of GLD-1. Since the last common ancestor of C. briggsae and C. elegans must have contained orthologs of 30 of 31 C. elegans sex determination genes, a change in the regulation of one or more of these genes might be responsible. Alternatively, since much of the regulation of C. elegans germline sex determination is by translational control, mutations in UTRs of mRNAs may result in new genes coming under the control of GLD-1 or another RNA sex determination gene regulator  will be important for the identification of C. briggsae–specific genes, analogous to fog-2, that are necessary for self-fertile hermaphroditism.In egulator . AdditioC. elegans sex determination genes were obtained from Wormbase . C. briggsae genomic sequence was obtained from The Sanger Institute  or the Genome Sequencing Center , and protein sequences were obtained from either Wormbase or Ensemble . Best BLAST orthologs of C. briggsae sex determination proteins were obtained using C. elegans sex determination protein sequences as queries against C. briggsae predicted proteins and six-frame translated C. briggsae genomic sequence. C. briggsae proteins obtained at an e-value cutoff of 1 × 10−50 reciprocal best hits were recovered for 26 of 31 C. elegans proteins. NOS-1 and XOL-1 orthologs were identified at an e-value cutoff of 1 × 10−20 and were also reciprocal best BLAST hits between species. In each case a single reciprocal best hit was identified for each component of the sex determination pathway with the exception of FBF-1 and FBF-2, which returned the same best BLAST hit, and FOG-2. Searches of the non-redundant National Center for Biotechnology Information protein database (GenBank CDS+PDB+SwissProt+PIR+WormPep) with full-length FOG-2 as query revealed only weak similarity to the F-box motif for non–C. elegans or –C. briggsae sequences. Using the highly diverged C-terminal end of FOG-2, including a portion of the Duf38/FTH, or the GLD-1 interaction region of FOG-2 as query did not reveal any hits below an e-value of 0.01 in C. elegans or C. briggsae other than FOG-2 and FTR-1.Protein coding sequences of cloned C. elegans FTR family members were identified using FOG-2 as a query against WormPep release 112. Each potential FTR was scanned for an N-terminal F-box motif and C-terminal Duf38/FTH domain using the hidden Markov models (HMMs) for each domain (HMMER 2.3.2) [C. briggsae FTR family members were identified using FOG-2 as a BLAST query and HMMs. In C. elegans, fog-2 (Y113G7B.5), ftr-1 (Y113G7B.4), CE35646 (Y113G7B.1), CE24144 (Y113G7B.3), CE23289 (Y113G7B.6), and CE23288 (Y113G7B.7) are closely related and tightly linked on Chromosome 5. CE35646 was not included in later analysis because of a divergent N-terminal structure.FTR family members are defined by the presence of an N-terminal F-box and C-terminal Duf38/FTH domain (FTR) . C. elegR 2.3.2) . SimilarC. remanei based on the currently sequence assembly . Our preliminary analysis suggests that closest FOG-2 homologs from C. remanei have diverged from C. elegans approximately to the same level as the FTR genes in C. briggsae. A comprehensive phylogenetic analysis to resolve the relationships between C. elegans, C. briggsae, and C. remanei FTR family members will await accurate C. remanei protein predictions and a complete C. remanei assembly.An FTR family also appears to be present and expanded in the obligate male/female species C. briggsae hit to each C. elegans FTR protein used in the phylogeny was included in order to identify any potential one-to-one orthologous pairs along the FOG-2 branch. Non-homologous N- and C-terminal extensions were trimmed, and extremely distant family members unlikely to be functional FOG-2 orthologs were excluded to avoid long branch attraction [Alignments were generated using CLUSTALW, and conserved residues were identified with the Lasergene MEGALIGN  package and Dialign ,72, whictraction . Phylogetraction .a/Ks calculation were generated using Se-Al  to modify CLUSTALW-aligned cDNA or predicted cDNA sequences, and all gaps and frame-shifted regions were removed. Sliding-window Ka and Ks estimates [Codon-restricted alignments for Kstimates  were genstimates , and codstimates   and C. briggsae (AF16) were obtained from the Caenorhabditis Genetics Center University of Minnesota, Minneapolis, Minnesota, United States. Cultures of both were maintained on Escherichia coli OP50 on NGM plates at 20 °C as previously described [C. elegans and C. briggsae essentially as described previously [gld-1 and fog-3 were generated by PCR amplification of cDNA with SP6 (5′) and T7 (3′) linkers, gel purified, sequenced, and used in RNA synthesis reaction using the appropriate Ambion kit . Double-stranded RNAs were injected at 0.5 mg/ml into young adult N2 animals and F1 progeny collected 12–48 h post injection and matured to 24 h post L4 stage before gonads were dissected, fixed, and stained to score for abnormal phenotypes.escribed . RNAi waeviously . Double-C. elegans and C. briggsae gonads were performed essentially as previously described with fixation in 3% formaldehyde, 80% methanol, and 100 mM dibasic potassium phosphate [Dissection, antibody, and DAPI staining of hosphate ,30. AffiGLD-1 and FOG-2 yeast two-hybrid binding assays were performed as previously described  with theFigure S1C. elegans and C. briggsae FTR genes (C. briggsae is in grey shade) is suggested by the phylogeny. The branch containing FOG-2 and FTR-1 is in bold. Tree is unrooted, and branch lengths are proportional to divergence. Bar represents 0.1 substitutions per site. Bootstrap support for separation of C. elegans and C. briggsae sequences is indicated at the node (black dot) and at each node for the C. elegans FOG-2 branch.A clear separation of (34.1 MB TIF).Click here for additional data file.Figure S2(A) FTR-1 and FTR family alignment. Residues identical to FTR-1 are shaded black, and residues identical between all FTR family members tested are shaded red. Average pairwise identity to FTR-1 is 48%.(B) FOG-2 and FTR family alignment. Residues identical to FOG-2 are shaded black, and residues identical between all FTR family members tested are shaded red. Average pairwise identity to FOG-2 is 22%.(15.6 MB TIF).Click here for additional data file.Table S1(59 KB PDF).Click here for additional data file.Table S2(59 KB PDF).Click here for additional data file.


The paca is a South American rodent with potential as a commercial food animal. We examined paca placenta as part of a wider effort to understand the reproductive biology of this species.Thirteen specimens between midgestation and term of pregnancy were studied by light and transmission electron microscopy.The placenta is divided into several lobes separated by interlobular trophoblast. Maternal arterial channels and fetal veins are found at the centre of each lobe. In the labyrinth, maternal blood flows through trophoblast-lined lacunae in close proximity to the fetal capillaries. The interhaemal barrier is of the haemomonochorial type with a single layer of syncytiotrophoblast. Caveolae occur in the apical membrane of the syncytiotrophoblast and recesses in the basal membrane, but there is no evidence of transtrophoblastic channels. The interlobular areas consist of cords of syncytiotrophoblast defining maternal blood channels that drain the labyrinth. Yolk sac endoderm covers much of the fetal surface of the placenta. The subplacenta comprises cytotrophoblast and syncytiotrophoblast. There are dilated intercellular spaces between the cytotrophoblasts and lacunae lined by syncytiotrophoblast. In the junctional zone between subplacenta and decidua, there are nests of multinucleated giant cells with vacuolated cytoplasm. The entire placenta rests on a pedicle of maternal tissue. An inverted yolk sac placenta is also present. The presence of small vesicles and tubules in the apical membrane of the yolk sac endoderm and larger vesicles in the supranuclear region suggest that the yolk sac placenta participates in maternal-fetal transfer of protein.The paca placenta closely resembles that of other hystricomorph rodents. The lobulated structure allows for a larger exchange area and the development of precocial young. Agouti paca, L) is a South American rodent that lives in forested habitats near water and feeds largely on fallen fruit. It is hunted for its meat, which is considered a delicacy, and is an important source of animal protein for rural populations. This has led to indiscriminate exploitation, resulting in a significant reduction in the population density of this species in Brazil.The paca . The experimental protocol was approved by the Bioethics Committee of the School of Veterinary Medicine, University of Sao Paulo.®, Fort Dodge, Campinas, S.P., Brazil; 8,000–24,000 IU/kg I.M.) and an analgetic . A detailed description of the anaesthesia and surgical procedures has been published elsewhere [The animals were sedated with azaperone  and given atropine (0.5 mg I.M.). Anaesthesia was induced with xylazine  and ketamine . Hemihysterectomy was then performed under aseptic conditions during inhalation anaesthesia with halothane . Postoperatively the animals were treated with benzyl penicillin and streptomycin . Sections were stained with haematoxylin and eosin, Masson's trichrome or Gomori's trichrome or by the periodic acid Schiff (PAS) reaction with haematoxylin as counterstain.Seven placentae were processed for transmission electron microscopy. Small samples were fixed in 2.5% glutaraldehyede in 0.1 M phosphate buffer, pH 7.4, washed in buffer and post-fixed in 1% osmium tetroxide . They were then dehydrated, washed with propylene oxide and embedded in Spurr's resin . 60 nm sections were made and stained with 2% uranyl acetate (5 minutes) and 0.5% lead citrate (10 minutes). The ultrastructural observations were made with a transmission electron microscope .The overall plan of the paca placenta is shown schematically in Figure The centres of the lobes, the labyrinth and the interlobular regions are clearly defined Figure . The cenThe most extensive portion of the lobe is the labyrinth. Due to close proximity between maternal and fetal blood vessels, it is the region where most maternal-fetal exchange takes place. The maternal blood spaces or lacunae are not lined by endothelium; they are defined by trophoblastic cell columns or cords. The cell columns are radially arranged as is apparent when the lobe is seen in cross section Figure . The synThe interlobular regions comprise cords of syncytiotrophoblast with abundant basophilic cytoplasm. These cords define maternal blood channels. The channels converge upon larger blood spaces, still without an endothelial lining, that receive blood from two or more adjacent lobes. Thus, each interlobular region is common to several lobes Figure . The intMost of the surface of the placental disk is covered by an epithelium formed by the endoderm of the parietal yolk sac Figure . An almoThe subplacenta is organized as folded lamellae of cytotrophoblasts supported on a thin layer of mesenchyme carrying fetal vessels Figure . The cytGroups of giant cells are found in the junctional zone between the subplacenta and decidua Figure . They arThe placenta is attached to the uterus by a placental pedicle made up of uterine tissue. In the upper region of this pedicle, a fine and discontinuous layer of connective tissue is interposed between the maternal tissue and fetal trophoblast. In the middle portion of the pedicle, a large number of vessels pass to or from the placenta. This region is characterized by dense fibres of connective tissue externally and of looser connective tissue around the vessels Figure . A layerThere is an inverted yolk sac placenta, which is attached to the fetal surface of the chorioallantoic placenta. Just before attachment it forms the fibrovascular ring Figure . The yolThe paca placenta is of the syncytial haemomonochorial type. A single trophoblast layer can be identified between the blood in the maternal blood spaces of the labyrinth and the endothelium of the fetal capillaries Figure . This trIn the interlobular areas, the syncytiotrophoblast bordering the maternal blood spaces has numerous microvilli Figure . The cytThe cells of the parietal yolk sac endoderm form a columnar epithelium Figure . These cThe cytotrophoblast layer is multilaminar with dilated intercellular spaces Figure . The cytIn the junctional zone between the decidua and the lateral aspect of the subplacenta, there are nests of multinucleated giant cells Figure . Their mThe apical surface of the endoderm cells has numerous microvilli of relatively uniform length Figure . Small vAs in other hystricomorph rodents ,4, the pIn the labyrinth, the trophoblast is bathed directly by maternal blood and is separated from the fetal capillaries by a single layer of syncytiotrophoblast. Thus the placental barrier is syncytial haemomonochorial, as in the guinea pig -9, chincIn the interlobular region, the trophoblast contained mitochondria and rough endoplasmic reticulum. The surface in contact with maternal blood bore numerous microvilli. The interlobular trophoblast is the functional equivalent of the spongy zone of murid rodents . In the Much of the surface of the placental disk is covered by the endoderm of the parietal yolk sac. This epithelium is largely columnar and rests on Reichert's membrane. Beneath the membrane are trophoblast cells that differ from those in other regions of the placenta in their larger size, rounder form and vacuolated appearance. An appropriate designation for these cells is spongiotrophoblast. It has been shown in the guinea pig that maternal protein can cross the spongiotrophoblast and Reichert's membrane. It can then pass into the uterine lumen through the intercellular spaces between the endoderm cells, as they are not sealed by tight junctions . In theoThe subplacenta is a unique feature of hystricomorph rodents . CharactWe found multinucleated giant cells in the junctional zone between the subplacenta and decidua. Intriguingly, the cytoplasm of these cells contained electron-dense granules reminiscent of those found in the subplacental syncytium. The cytoplasm of the giant cells had areas of low electron density, a feature also shared by the subplacental syncytium. These cells were PAS-positive and may store glycogen or glycoprotein.The placenta of paca is attached to the uterus by a prominent structure, formed largely of maternal tissue, that we have denoted the placental pedicle. It was first described by Strahl  and nameLike other hystricomorph rodents, paca has an inverted yolk sac placenta that persists until term. This visceral yolk sac displays folds and complex villi. The numerous digitiform projections are sometimes branched. They consist of a mesenchymal axis covered by a simple columnar epithelium of endodermal cells. The cells seem to have a high level of endocytotic activity. Similar characteristics are found in the yolk sac endoderm of the guinea pig , chinchiIn conclusion, the placenta of the paca conforms to patterns previously described for hystricomorph rodents ,14. CommRecently it was argued that more attention should be given to the hystricomorph rodents as models in human medicine. They bear a closer genetic similarity to humans than do murid rodents, such as the mouse and rat, because the latter have undergone a very high rate of gene mutation . Since t


The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression. E. coli is the most popular host for the expression of heterologous proteins but its simplified cell organization can be limiting for the expression of correctly folded recombinant proteins. No bioinformatic tools can predict if a construct will be expressed soluble in bacteria and, therefore, time-consuming cloning steps and expression optimization tests must be considered.The possibility to produce recombinant proteins instead of recovering the native molecules offers the double advantage of higher yields and of a simplified purification protocol using affinity chromatography. At least half a dozen of the purification tags that have been proposed so far are routinely fused to the target proteins and used to perform affinity purification. In most of the cases the affinity purification protocols are effective. However, the costs of the resins and proteases necessary to remove the tags can become a limiting factor. Furthermore, the different requirements for the chromatography steps and proteolysis make difficult to conceive automatic systems for obtaining purified homogeneous protein. As an alternative, we showed that the fusion of a target protein with a thermostable partner can be purified to homogeneity by heating . BecauseWe do not expect that heated proteins recover the native structural features but it is a common simplification to assume that a soluble protein is correctly folded and companies commercialize vectors with tags that "improve the solubility". Completely underestimated is the fact that a very soluble fusion partner can keep in solution unfolded target proteins. The work of the group around Travé showed the false results generated using fusion proteins and suggested a method for the evaluation of the aggregation state ,3 and toTherefore, methods would be envisaged that combine the purification to the selection for the correct folding. An important contribution in this direction is the recent paper published by Greg Winter's group .The authors  describeFunctional genomic relies on the possibility of screening fast and efficiently large number of clones for their expression and correct folding. Several approaches have been suggested over the past years. An indirect method considers marker genes activated by misfolding  to discrin vivo, directly and exclusively, for the class of conventional antibodies able to fold in the cytoplasm [The elegance of the method described by Winter and co-workers relies on the use of a ligand that recognizes only the folded state of the protein to purify: the quality control is inclusive in the affinity purification. Furthermore, the phage-display format allows for the identification of the corresponding clone. As pointed out by the authors, such an approach is limited to those cases for which a conformation-dependent bait is available or, at least, a reliable method exists to discriminate between native and aggregated proteins. The logic of the experiment reminds me to the protocol used to select ytoplasm .in vivo [in vivo protein quality control.It is difficult to envisage a method applicable to all protein classes for selecting constructs that will express correctly folded proteins. Nevertheless, it is still possible to improve the yield of recombinant proteins that tend to aggregate. At least part of the unfolded proteins is not definitely trapped in aggregates. Re-solubilisation from bacterial inclusion bodies happens in vivo , the invin vivo ,11 and win vivo . Our unpThe recombinant expression of proteins often induces the formation of soluble aggregates and several of such aggregates conserve sufficient features for being recovered by affinity chromatography. The simplification of having considered a purified soluble protein as a protein in native state has generated false results . TherefoFinally, the heat-selection can provide useful insights about the molecular features involved in the (re)-folding/disaggregation mechanisms.


For the past several decades, retrovirologists from around the world have gathered in late May at the Cold Spring Harbor Laboratories in New York to present their studies in formal talks and posters, and to discuss their ongoing research informally at the bar or on the beach. As organizers of the 2004 Cold Spring Harbor Retroviruses Conference, we have been asked by the editors of Retrovirology to prepare a review of the meeting for publication on-line. Our goal in this review is not to provide a detailed description of data presented at the meeting but rather to highlight some of the significant developments reported this year. The review is structured in a manner that parallels the organization of the meeting; beginning with the entry phase of the replication cycle, proceeding with post-entry events, assembly and release, integration, reverse transcription, pathogenesis/host factors, RNA-related events  and finishing with antivirals. While the most striking developments this year involved post-entry events and assembly/release, significant progress was made towards elucidating a number of aspects of the retroviral replication cycle. They also suggested that Env-mediated impairment of GLUT-1 function might contribute to the emergence of preleukemic T cells with new selective advantages  assemble at the plasma membrane. However, it has long been recognized that in some cases  assembly and budding take place at an intracellular compartment. Several groups have recently demonstrated that this compartment is the late endosome or multivesicular body (MVB), and it now appears that while the plasma membrane likely represents the predominant site of assembly for these viruses, Gag can also target and assemble in an endosomal compartment in a variety of cell types. M. Resh's lab (Perlman and Resh) used the tetracyteine/biarsenical live-cell labeling system reported by Gaietta et al.  to folloH. Wang from L. Mansky's group observed that HTLV-1 assembly, like that of HIV-1, can take place in the MVB. Interestingly, while in HeLa cells HTLV-1 assembly appears to occur primarily at the plasma membrane, mutational disruption of the Pro-Thr-Ala-Pro (PTAP) late domain results in accumulation of virus particles in the MVB, consistent with published results . T. Hop2) plays a role in Gag targeting. This lipid is of interest since it is involved in the trafficking of a variety of cellular proteins that, like many retroviral Gag proteins, contain a basic membrane binding domain. Furthermore, work from A. Rein's lab [in vitro, HIV-1 Gag binds molecules structurally related to phosphoinositides. A. Ono used enzymatic approaches to perturb cellular PIP2 levels in virus-expressing cells and observed that Gag targeting and virus assembly were shifted from the plasma membrane to intracellular compartments. These results indicate that cellular PIP2 plays a role in directing Gag to the plasma membrane.A study from the Freed lab (Ono and Freed) examined the possibility that the phosphoinositide phosphatidylinositol--bisphosphate Pin's lab  has showWork from J. Lingappa's lab previously suggested the involvement of the cellular RNase L inhibitor HP68 in HIV-1 assembly . This woNovel findings relating to the regulation of foamy virus (FV) release were reported by the Lindemann lab. FVs are unusual among retroviruses in that Env glycoprotein expression plays a critical role in particle release. The leader peptide of FV Env, which is generated by a cleavage event during Env trafficking to the plasma membrane and is incorporated into particles, seems to play an important role in the budding process. Lindemann et al. reported the identification of ubiquitylated forms of the leader peptide; suppression of these ubiquitylated forms markedly stimulated subviral particle release. These results suggest that ubiquitylation of the FV Env leader peptide modulates the ratio of particle vs. subviral particle budding.Progress continues to be made in visualizing retrovirus assembly and release in real time in living cells. In addition to use of the tetracyteine/biarsenical live-cell labeling system described above, B. Muller from H.-G. Krausslich's lab discussed the development of an infectious HIV-1 derivative containing a GFP insert near the C-terminus of MA. This derivative produces particles with wild-type morphology; however, release kinetics are impaired. While the Gag-GFP virus is poorly infectious, infectivity can be restored upon coexpression with wild-type HIV-1. This Gag-GFP HIV-1 derivative should be useful for both assembly/release and post-entry studies. In a presentation from the labs of V. Vogt and W. Webb, D. Larson described the use of correlated fluorescence microscopy and scanning electron microscopy to visualize Rous sarcoma virus (RSV) budding in real time using a GagGFP derivative.Major advances have been made in the past three years in elucidating the host cell machinery required for the release of retrovirus particles from infected cells ,30. It iIn yeast, ESCRT-I contains three protein components: Vps23 (the yeast homolog of Tsg101), Vps28 and Vps37. In mammalian cells, only the Vps23 and Vps28 homologs had been identified. At this year's meeting, M. Stuchell from W. Sundquist's lab reported the cloning of human Vps37. As in yeast, human Vps37 binds Tsg101 and is present along with Tsg101 and Vps28 in a high molecular weight ESCRT-I complex. Much of this work has recently been published , as haveM. Palmarini's lab  described a novel type of retroviral interference that operates at the level of virus assembly and release. The sheep genome harbors a number of copies of endogenous retroviruses closely related to the pathogenic exogenous β-retrovirus Jaagsiekte sheep retrovirus (JSRV). One of these endogenous retroviruses (enJS56A1) displays a defect in assembly/release. EM analysis indicates that enJS56A1 particles form large perinuclear aggregates; these appear to trap JSRV particles intracellularly when enJS56A1 and JSRV are coexpressed. The authors speculate that enJS56A1 may have helped protect sheep from infection with related, exogenous retroviruses during evolution. Much of this study has recently been published .It has been known for many years that several retroviruses express two forms of Gag: a conventional version and a larger, glycosylated form referred to as glycoGag. GlycoGag is synthesized using an alternative, upstream initiation codon, and unlike its smaller counterpart it is transported to the plasma membrane through the secretory pathway. H. Fan's lab  reported at this year's meeting that a packaging cell line that expresses Gag without glycoGag produces tube-shaped particles at the cell surface. This apparent assembly defect was corrected by coexpression of glycoGag. The expression of glycoGag increased both virus yield and infectivity. These results suggest that glycoGag may function, in a manner that remains to be elucidated, in regulating proper virus assembly and release.Retroviral preintegration complexes (PICs) and the integrase (IN) enzyme itself, interact with a variety of host factors during nuclear import of the PIC and integration of the newly synthesized viral DNA into the host cell chromosome. One such host factor, lens epithelium-derived growth factor (LEDGF/p75), was recently reported to bind HIV-1 IN ,38. SeveA number of presentations focused on the target site specificity of retroviral (or retrotransposon) integration. Different retroviruses and retroelements display strong preferences in selecting their target sites in the genomes of their host cells (for review see ). For exM. Katzman's lab  reported an interesting RSV IN mutant that displayed enhanced 3'-end processing activity but impaired DNA joining. The authors speculated that RSV IN has evolved suboptimal processing activity to allow it to catalyze DNA joining.R. Craigie's lab previously reported that a protein termed "barrier-to-autointegration factor" (BAF) is a component of the MLV preintegration complex and that this factor enhances intermolecular integration reactions and blocks intramolecular integration (autointegration) . At thisin vitro assay that models minus-strand transfer, NC alone is able to catalyze the removal of small 5' terminal genomic RNA fragments, which remain annealed to a minus-strand strong-stop DNA. Strand transfer product increased with increasing NC, and in the presence of NC, strand transfer product was generated even when reverse transcription was catalyzed by an RNase H-deficient RT. The NC zinc fingers appeared to be critical for this activity. These results suggest an important role for NC nucleic acid chaperone activity in removing terminal RNA fragments annealed to minus-strand strong-stop DNA following primary cleavage by RNase H. By examining reverse transcription in infected cells, R. Gorelick's lab  observed that mutations in the first zinc finger of NC strongly interfered with the progression of reverse transcription and impaired virus infectivity. The authors speculated that reduced binding of NC to the viral DNA allows cellular enzymes (nucleases and ligases) to modify the viral DNA ends thereby interfering with integration.A number of studies over the years have demonstrated that retroviral NC proteins possess nucleic acid chaperone activity that plays an important role in various aspects of reverse transcription . Severalys3. The selectivity for particular tRNAs results from interactions between the tRNA and the PBS and it has been proposed by B. Berkhout and colleagues that a second motif in the viral RNA, termed the primer-activation signal (PAS), also forms specific contacts with the tRNA. Berkhout's lab  reported at this year's meeting that, by mutagenesis of the PBS and PAS, HIV-1 primer utilization could be shifted from tRNAlys3 to tRNAlys1,2. Such mutants replicated poorly but could adapt during long-term passaging. In one case, adaptation evidently occurred through optimization of the putative PAS. Interestingly, a single amino acid change in the RNase H domain of RT also arose during adaptation, suggesting a possible role for RT in selective primer utilization.Reverse transcription is initiated by a host cell-derived tRNA bound to the primer binding site (PBS) near the 5' end of the viral genome. Retroviruses are selective in their utilization of host tRNAs; HIV-1, for example, specifically primes reverse transcription with a tRNAWhile PCR techniques allow the progression of viral DNA synthesis to be monitored post-infection, it has not been possible to follow reverse transcription in a strand-specific fashion in infected cells. D. Thomas from V. Pathak's lab reported the development of a strand-specific amplification assay that uses so-called "padlock" probes – long single-stranded oligonucleotides that hybridize with their target sequence simultaneously at their 5' and 3' ends. Following hybridization, the ends of the padlock probes are ligated to form circles that are amplified and detected by real-time PCR. This assay, which allows specific steps in reverse transcription to be measured quantitatively in a strand-specific manner, should be very useful for addressing a number of questions regarding the kinetics of reverse transcription and the efficiency of this process under a variety of conditions.in vivo. The authors monitored genetic variation in cohorts of drug-naïve and drug-resistant patients by analyzing individual pro-pol sequences. In both drug-naïve and drug-resistant patients, they observed little change in virus population structure over several years, implying that the replicating population is relatively large. Interestingly, recombination rates appeared to be very high regardless of levels of viremia, suggesting the presence of a substantial number of multiply infected cells even at low viral loads. The results emphasize the importance of recombination in generating viral diversity in vivo.F. Maldarelli described the results of studies conducted with S. Palmer, J. Coffin and colleagues aimed at characterizing the evolution of HIV-1 populations The keynote speaker at this year's meeting was Neal Copeland, who presented important findings from his and Nancy Jenkins' lab regarding the use of high through-put analysis to map retroviral integration sites in tumors induced by MLVs. This rapidly developing approach is being used by a number of groups to elucidate cancer gene pathways -46, and I, which is targeted to the endoplasmic reticulum/Golgi of infected cells and decreases MHC class I trafficking to the cell surface. R. Fukomoto from G. Franchini's lab presented data that p12I decreases activation of the transcription factor NFATI in T cells by binding to LAT and inhibiting T-cell receptor signaling. In contrast, M. Lairmore presented data that p12I expression in Jurkat cells results in ~20-fold activation of NFAT-dependent gene expression in a calcium-dependent manner (Kim and Lairmore). These authors also demonstrated that p12I acts in the endoplasmic reticulum to activate calcium-mediated T-cell activation during the early stages of infection, apparently through an interaction with calcineurin. These studies suggest a prominent role for p12I in common T cell activation pathways critical to the establishment of a persistent infection. Another protein, p13II, which was described by V. Ciminale , is localized to the inner mitochondrial membrane and induces changes in Ca2+ and K+ permeability. In culture, p13II reverses the morphological transformation of rat embryo fibroblasts expressing c-Myc and Ha-Ras and decreases their ability to form tumors in nude mice. There was speculation that HTLV-I encodes p13II to counteract the growth-inducing properties of the other viral accessory proteins (such as Tax) that are required for establishment of infection, thereby allowing the virus to persist in infected individuals.In contrast to the majority of leukemia-inducing retroviruses in non-human species, most of which cause cancer by insertional mutagenesis, HTLV-I encodes accessory proteins, such as Tax, with known transforming activity. There were several talks at the meeting describing new accessory proteins that may also play a role in cell regulation. Two talks focused on the protein p12In a theme that echoed in several of the simple retrovirus talks, V. Armbruester from the Mueller-Lantzsch laboratory reported on a novel protein generated by alternative splicing of the envelope gene of the HERV-K endogenous retrovirus. The transcript for this protein, np9, is highly expressed in mammary carcinomas and germ cells, and the gene product binds to the LNX protein, which is a ligand of Numb and targets it for proteasomal degradation. Since LNX/Numb/Notch is a known transformation pathway in tumors, Armbruester speculated that np9 may play a role in tumorigenesis by sequestering LNX, thereby stabilizing Numb.This session surveyed new findings on retroviral splicing, nuclear retention and export, translation and packaging. J. Madsen and M. Stoltzfus evaluated the role of an exonic splicing silencer (ESS) on HIV-1 replication in cultured T cells. An HIV-1 mutant with ESS substitutions displays a replication-defective phenotype that correlates with increased viral RNA splicing. This mutant was subjected to long-term passage and the viruses that emerged contained second-site reversions in splice sites flanking the exon containing the mutated ESS. Stoltzfus speculated that strains that do not contain the ESS maintain balanced expression of their viral genome by a novel, unknown mechanism.A. Lever's lab  described confocal microscopy and fluorescence resonance energy transfer (FRET) analysis of the interaction between HIV-1 Gag and its cognate genomic RNA. Using biotinylated probes to the full-length, unspliced RNA, he described an initial perinuclear co-localization between Gag and genomic RNA that subsequently shifted to the plasma membrane. Mutation of the Ψ packaging signal partially disrupted the perinuclear and plasma membrane colocalization.env gene. The gene was designated rem, (for RNA export protein of MMTV RNA). Its product stimulated nucleocytoplasmic transport of the unspliced MMTV transcript, in a manner similar that of the Rec protein of HERV-K [gag, pol, and env). The Dudley group suggested that since MMTV exhibits a complex genetic structure it should be reclassified as a complex retrovirus.J. Dudley's lab  described a new MMTV gene that encodes a viral RNA transport protein generated by alternative splicing of the f HERV-K ,48. ThisK. Boris-Lawrie  presented genetic and biochemical data showing that the R-U5 region of SNV RNA adopts a stem-loop structure that stimulates cap-dependent translation. RNA affinity and proteomic analysis showed that RNA helicase A (RHA) bound to wild-type RNA but not to mutants containing substitutions in this structure. RHA interaction with SNV R-U5 stimulated translation of unspliced HIV-1 reporter mRNA. Boris-Lawrie speculated that this could occur by rearrangement of intramolecular RNA interactions that disrupt the packaging signal, thus facilitating mRNA translation.In vivo, only a fraction of HTLV-infected cells actively expresses viral RNA, leading to speculation that the virus negatively regulates gene expression. P. Green  described a new role for HTLV-1 and -II accessory proteins p30 and p28, respectively, in negatively regulating virus production during chronic infection. Tax/Rex expression was inhibited upon ectopic expression of p28 by a mechanism that involved nuclear retention of the mRNA. Analysis of RNAs bearing a luciferase reporter gene showed that the 3' splice junction was sufficient to confer this nuclear retention. From this work and the studies from the Franchini and Lairmore labs described above, it is clear that HTLV-1 has evolved to regulate its expression in infected cells, thereby evading immune recognition and promoting viral persistence.As in previous years, there was a strong emphasis on the development of antiviral agents that interfere with reverse transcription. However, noteworthy progress was also reported in efforts to target a number of additional steps in the replication cycle.in vitro assays to examine the effect of RT polymerase inhibitors on RNase H activity. They observed that NNRTIs actually increased RNase H activity; structural studies suggested that this enhancement was due to greater accessibility of the DNA/RNA duplex by RNase H. Although a diketo acid RNase H inhibitor displayed decreased potency in the presence of an NNRTI, the diketo acid and NNRTI synergistically inhibited reverse transcription overall.S. Sarafianos reported data from E. Arnold's lab  on the structure of HIV-1 RT in a complex with an RNase H inhibitor. Interestingly, the binding site of the compound is quite distant (>40 Angstroms) from the RNase H active site and partially overlaps the NNRTI binding pocket. These results raise the intriguing possibility that compounds could be designed that simultaneously act as RNase H inhibitors and as NNRTIs. M. Miller's lab  performed C. Wild, E. Freed, and colleagues, and independently C. Aiken's lab, previously reported that a dimethyl succinyl betulinic acid derivative (referred to as PA-457 or DSB) potently blocks HIV-1 infectivity by specifically disrupting the cleavage of the CA precursor (composed of CA and spacer peptide SP1) to mature CA ,50. The A. Lever's lab  described their efforts to inhibit HIV-1 replication using oligonucleotides that target the viral genome. The authors observed that oligos targeting the packaging signal  disrupt Gag binding and reduced virus infectivity. T. Murakami and coworkers reported the development of an orally bioavailable compound that binds the HIV-1 chemokine coreceptor CXCR4. In culture, the compound potently inhibits infection by HIV-1 isolates that use CXCR4 as a coreceptor, but, as expected, do not block infection by strains that exclusively use CCR5 as a coreceptor. The compound suppressed HIV-1 infection in the hu-PBL-SCID mouse model. The results of this study suggest that this CXCR4 antagonist could potentially be an effective drug in infected humans.It has been suggested that most virus originating in the central nervous system (CNS) derives from long-lived cells  that would continue to produce virus for a significant period of time after the initiation of antiretroviral therapy. According to this model, CNS-derived virus should decay more slowly following the onset of therapy relative to virus derived from the blood. In the last presentation of the conference, data were presented from R. Swanstrom's lab  obtained from a study of HIV-1 population dynamics in cerebrospinal fluid (CSF) immediately following the initiation of antiretroviral therapy. Virus isolates in the CSF apparently derive from both the CNS and the blood plasma. Interestingly, using heteroduplex tracking assays, the authors observed that within the first several days following the initiation of therapy CNS-derived isolates in the CSF decline with similar kinetics to isolates shared with the blood, suggesting that virus from both compartments is produced by cells with a short life span.


Normalization is a basic step in microarray data analysis. A proper normalization procedure ensures that the intensity ratios provide meaningful measures of relative expression values.We propose a robust semiparametric method in a two-way semi-linear model (TW-SLM) for normalization of cDNA microarray data. This method does not make the usual assumptions underlying some of the existing methods. For example, it does not assume that: (i) the percentage of differentially expressed genes is small; or (ii) the numbers of up- and down-regulated genes are about the same, as required in the LOWESS normalization method. We conduct simulation studies to evaluate the proposed method and use a real data set from a specially designed microarray experiment to compare the performance of the proposed method with that of the LOWESS normalization approach.The simulation results show that the proposed method performs better than the LOWESS normalization method in terms of mean square errors for estimated gene effects. The results of analysis of the real data set also show that the proposed method yields more consistent results between the direct and the indirect comparisons and also can detect more differentially expressed genes than the LOWESS method.Our simulation studies and the real data example indicate that the proposed robust TW-SLM method works at least as well as the LOWESS method and works better when the underlying assumptions for the LOWESS method are not satisfied. Therefore, it is a powerful alternative to the existing normalization methods. Microarray technology has become a useful tool for quantitatively monitoring gene expression patterns and has been widely used in functional genomics ,2. In a A basic question in analyzing cDNA microarray data is normalization, the purpose of which is to remove systematic bias in the observed expression values by establishing a normalization curve across the whole dynamic range. A proper normalization method ensures that the normalized intensity ratios provide meaningful measures of relative expression levels. Normalization is needed because many factors, including different efficiency of dye incorporation, difference in the amount of RNA labeled between the two channels, uneven hybridizations, difference in the printing pin heads, among others, may cause bias in the observed expression values. Therefore, proper normalization is a critical component in the analysis of microarray data and can have important impact on higher level analysis such as detection of differentially expression genes, classification, and cluster analysis.invariant genes that are likely to be constantly expressed and then carrying out LOWESS normalization using this set of genes. But as pointed out by Tseng et al., selected invariant genes may not cover the whole dynamic range of the expression values, and extrapolation is needed to fill in the gaps that are not covered by the invariant genes. Kepler et al. [Many normalization methods have been proposed in the literature. The earliest normalization method for cDNA microarray data goes back to Chen et al.  who propr et al.  also firr et al.  proposedr et al.  used arrr et al.  comparedr et al.  used a mr et al.  considerr et al.  generalir et al.  proposedIt is well known that the least squares method is not resistant to outliers which arise often in cDNA microarray experiments because of many sources of variations. In this paper, we propose a robust method for normalization in the framework of the TW-SLM. We conduct simulation studies and use a real cDNA microarray data set to compare the proposed method with the LOWESS normalization method.jth gene is calculated as the following:Simulation was conducted to compare the mean square errors (MSE) and biases of estimated gene expression levels between the proposed robust TW-SLM and LOWESS normalization methods, between the proposed method and the TW-SLM using OLS. The MSE for the , where N is the total number of replicates for each simulation, J is the number of unique genes, βj is the true gene expression level  for gene j,  is the estimated value for βj,  is the mean of  for N replicates, j = 1, 2,..., J, where J is the total number of genes. The data simulation procedure is based on the method proposed by Balagurunathan et al. [that is, n et al. . In eachj using the exponential distribution with the mean of 3,000, i.e. Ij ~ exp(λ = 1/3000), for j = 1,..., J;1. Simulate true signal intensity for each gene αrj and αgj, respectively, then the fluorescent intensity on the two channels can be generated by the normal distribution with mean Ij and standard deviations αrjIj and αgjIj for the red channel and the green channel, respectively. Let Rj and Gj represent simulated fluorescent intensity for the Cy5 channel and the Cy3 channel for gene j, respectively;2. Simulate fluorescent intensity for the Cy5 channel and the Cy3 channel with the normal distribution, respectively. Suppose the coefficients of variation for intensity in the Cy5 channel and the Cy3 channel are γ × 100% genes are differentially expressed in the whole simulated gene set, then the ratio of the expression level for gene j can be generated by tj = 10b ±with b ~ Beta. The sign ± will determine if the gene is up- or down-regulated. The probability of the up-regulated genes within those γ × 100% differentially expressed genes is given as an input parameter. For the genes that are not differentially expressed, the b takes value zero;3. Simulate differentially expressed genes. Suppose tj into signal intensity of gene j. The Rj and Gj will be adjusted by adding the simulated expression ratio tj through the following formulae:  for j = 1,..., J;4. Incorporate the 5. Simulate a fluorescent system with the imperfect response characteristics. Due to various reasons, such as the unequal amount of mRNA for the two channels, different labeling efficiencies, or uneven laser powers at the scanning stage , actual  and , respectively. So four parameter values need to be determined for each channel before simulation. Different parameter values in the two channels will control the shape of the ratio vs. signal intensity plots (R-I plots);to distort the response characteristic functions of observed fluorescent intensity for the two channels, which are expressed as αbr and αbg for the Cy5 channel and the Cy3 channel, respectively. These two parameters are the ratios between the mean and the standard deviations of background noise for the two channels, respectively. Simulated signal intensity for the two channels,  and , are adjusted by subtracting background noise in each channel. Let  and  still denote background adjusted signal intensity for the two channels;6. Simulate background noise for each channel. The mean of background noise is determined by one input parameter: the signal to noise ratio (SNR) and the true mean of signal. The SNR is the ratio between the true mean of the signal and the true mean of background noise. The SNR controls variability of background noise. The normal distribution with a given mean value is used in simulating background noise. Variance of background noise will be controlled by the input parameters 7. Add noise to the signal intensity for each channel. Finally, the signal intensity of each channel is generated by, where α1 ~ U , α2 ~ U , α3 ~ U , α4 ~ U . The a1, b1, c1, d1, a2, b2, c2, d2 are given as input parameters to control variability of fluorescent signal intensity.with We simulated two situations, one is the no-dye bias case and another one is the shape case (dye bias exists). R-I plots of twelve blocks on one slide for two simulated cases are shown in Figures The trend of MSEs and biases of estimated gene expression levels are similar between the robust TW-SLM and the LOWESS normalization methods across different levels of the ratios between the up-regulated genes and the down-regulated genes. This trend also exists in the extreme case. We present the results of the following two scenarios: (a) a 9:1 ratio between the number of the up-regulated genes and that of the down-regulated genes and, (b) the extreme case. Tables Comparing the different robust weight functions, means of MSEs are slightly smaller using Tukey's weight function than that using Huber's weight function. These results are observed across different percentage levels of differentially expressed genes. Biases for estimated gene expression levels distributed similarly between the proposed method and the LOWESS normalization method. But the ranges of the biases for the proposed method are smaller than those of the LOWESS normalization method and the TW-SLM using OLS, respectively. These observations are true in both simulated situations.The extreme case is an example where the proposed method does better than the LOWESS method Tables  and 7. EThe distributions of MSEs and biases between the TW-SLM using OLS and the LOWESS method are similar for cases where there is a relatively small percentage of differentially expressed genes. However, the TW-SLM with OLS performs better than the LOWESS when a larger proportion of genes are differentially expressed. It appears that the more deviation from the two assumptions required by the LOWESS, the better the TW-SLM performs. This trend is consistent with findings in our previous work .In this section, a real data set was analyzed to compare consistency of the LOWESS normalization method and the proposed robust TW-SLM method. A collection of human placenta cDNAs comprising 7,042 clones was identified and used as the probe set for cDNA microarray fabrication in this study .in vitro transcription from a pool of cDNAs in equal amount comprising the entire probe set (PS); (ii) the "Universal Human Reference RNA" from Stratagene, a pool of RNAs derived from 10 different cell lines; and (iii) human full-term placenta RNA. The original goal of the study was to evaluate the performance of the PS RNA as a reference RNA in comparison with that of Stratagene's universal reference RNA.Three kinds of RNA samples were used which include: (i) a common reference RNA obtained by In this study, the Universal Human Reference RNA and the human placenta RNA were treated as two experimental samples. The PS RNA was used as the reference against which the two other bio-samples were compared. In the simple direct comparison, gene expression values were obtained through direct hybridizations between the human placenta RNA and the Universal Human Reference RNA. In the indirect comparison using the PS set as the common reference, hybridizations were performed between the human placenta RNA and the PS reference RNA, and between the Universal Human Reference RNA and the PS reference RNA. The design of this experiment is depicted in Figure After hybridization, slides were scanned with the Axon instruments 4000B scanner. The 633 and 532 lasers are used for excitation of the Cy5 and Cy3 fluorophores, respectively. For each of the three types of hybridizations , there are four slides, including two dye-swapped slides. Each clone was printed three times on different blocks on each slide. Background adjusted medians for the Cy5 and Cy3 channels were used as expression levels. We removed negative controls including "Human Cot1", "PolyA" and "Empty" in the analysis.To evaluate the proposed method, we compare it with the LOWESS method by examining which method produces more consistent results between the direct comparison and the indirect comparison of human placenta and universal human reference RNA samples as described above  in the direct comparison and about 61% (1713/2791) in the indirect comparison.We used 10-5 in the direct comparison and 1,045 in the indirect comparison. The number of overlapping genes with p-value less than 10-5 is 467, which is around 32% (467/1447) in the direct comparison and about 44% (467/1045) in the indirect comparison. It is clear that the proposed method performs more consistent between the direct comparison and the indirect comparison.In comparison, using the LOWESS normalization and the t-tests, there are 1,447 genes with p-value less than 10We also examined overlap between the LOWESS and robust TW-SLM methods for the two comparisons. In the direct comparison, about 79% (1141/1447) of the genes found to be significant based on the LOWESS method are also found to be significant based on the robust TW-SLM method. But they only account for about 40% (1141/2907) of the significant genes detected based on the robust TW-SLM method. In the indirect comparison, about 71% (738/1045) of the significant genes based on the LOWESS method are also found to be significant based on the robust TW-SLM method. But they only account for about 26% (738/2791) significant genes detected based on the robust TW-SLM method.In our analysis, we used background adjusted intensity values. How to adjust background is an important issue in microarray data analysis. To evaluate if background affects our conclusions, we repeated the comparison analysis without adjusting background for the intensity values in both channels, the results are presented in Tables Therefore, the robust TW-SLM method yields more consistent results between the direct comparison and the indirect comparison with the human placenta and the universal human reference RNA samples. In addition, the robust TW-SLM method detects more significant genes for a given cutoff p-value. This makes sense biologically because most of the 7,042 genes specifically discovered from human placenta are expected to have differential expressions relative to the universal reference RNAs. We would expect that the similar comparison results will be got if we compare the TW-SLM using OLS or Huber's weight function with the LOWESS method because the normalization curves for the TW-SLMs  are similar, but all these three curves are different from the LOWESS normalization curve Figure .βj as the thread linking these slides. In addition, in the proposed method, the normalization curves φi and gene effects βj are estimated simultaneously. With this approach, there is no need to assume that the percentage of genes with differential expression levels is small or the expression levels of up- and down-regulated genes are symmetric, or when one of these assumptions is not satisfied, to use dye-swap normalization, which in turn requires the assumption that the two normalization curves are symmetric.  An underlying condition required for the proposed method is independence of arrays, which is satisfied in a typical microarray experiment. Further theoretical conditions for the TW-SLM can be found in the paper by Huang et al. [We have proposed a robust TW-SLM normalization method for cDNA microarray data. It is interesting to compare the proposed normalization method with the existing methods, such as the widely used LOWESS normalization proposed by Yang et al. (2001)  and furtg et al. .We have only considered the proposed robust TW-SLM method for the simple direct comparison design described in the Methods section. We can easily extend the method to more complicated designs. For example, we can adapt the proposed robust method to the TW-SLM that accommodates the design where a gene is printed multiple times. Such a design is helpful for improving the precision and for assessing the quality of an array using the coefficient of variation . We canR package which is available from the corresponding authors.In our simulation studies, the proposed method performs better than the LOWESS normalization method in terms of MSEs of estimated gene effects in the simulation models we considered. Analysis of the probe set reference data set  shows thn be the number of slides, and J be the number of genes in the study. Let Rij represent background corrected signal intensity from the Cy5 channel and Gij the background corrected signal intensity from the Cy3 channel, and let yij = log2(Rij/Gij), xij = (1/2) log2(Rij × Gij), for gene j on slide i. We assume that there is only one spot for each gene on each slide. The TW-SLM [We first describe the TW-SLM. For simplicity, we focus on the case of comparing two cell populations, in which two cDNA samples from the respective cell populations are competitively hybridized on the same array. Let e TW-SLM  isyij = φi (xij) + βj + ∈ij, i = 1,..., n, j = 1,..., J     (1)φi which is the intensity dependent normalization curve for slide i, the second component is βj which represents the relative expression value of the jth gene after normalization, the last one is the residual error term. Let  be a robust estimator of the ith normalization curve φi based on this model described above. The normalized data areIn this model, the observed log intensity ratio is decomposed into three components. The first component is φi and βj in the TW-SLM. However, it is well known that least squares estimates are not robust against outliers which often arise in microarray experiments. Therefore, we propose to use the robust method [φi and βj. This is done by minimizing the objective functionHuang et al. 2004)  consider04  consiρ is an appropriately chosen function for robust estimation, λ is the collection of the coefficients in the spline representations of φi described below, σ is the scale parameter, and α is a constant to be described below. We note here that estimation of φi, βj are done jointly and uses data from all the arrays. This is different from the LOWESS normalization method in which estimation of normalization curves are done array by array.where ρ functions: Huber's ρ function and Tukey's biweight function. Huber's ρ function isWe consider two Tukey's biweight function isρ, ψ and χ, will be used repeatedly in the description of the algorithm below. They are defined asTwo other usefull functions derived from ψ(x) = ρ'(x), χ(x) = xψ(x) - ρ(x).     (4)H = 1.345 and k = 4.685. The influence of the choice of these constants on normalization methods is beyond the scope of this study.The expressions of these functions are given in the Appendix. We choose commonly used constants in the literature for Huber's and Tukey's functions, i.e., φi. Specifically, let b1,..., bK be K B-spline basis functions. We approximate φi byWe use the cubic B-splines ,22 to apb(x) = ,..., bK(x))' and λi = '.where We estimate the parameters in model (1) by minimizing objective function (3) using an iterative procedure. Two steps, a location step and a scale step, will be used in the computation.We use the following vector and matrix notations in describing the location step:Bi = (b(xi1), b(xi2),..., b(xiJ))',i y= '.Letand leti = 1,..., n. Given the scale parameter σ,  and  satisfy the equations:for  and  because of identifiability requirement in the TW-SLM. We can solve these equations iteratively to obtain  and . The derivations of these equations are given in the Appendix.where σ isAccording to Huber's proposal , the estrij = yij - b'(xij) , and N is the total number of observations in the data set. In general, equation (8) does not have an explicit solution. So we use the following updating equation to compute the estimated scale parameter σ,where , we used the constant suggested by Huber [In order to obtain the consistent scale estimator at the normal distribution and obtain the classic estimates when using the least squares objective function, i.e., by Huber ,EΦ denotes expectation with respect to the standard normal distribution function Φ.where The procedure described above is called an iterative reweighted least squares (IWLS) algorithm that is used in many non-least squares estimation problems. The implementation of the IWLS algorithm can be carried out using the following steps: for j = 1,..., J and σ(0) = 1, , for i = 1,..., n, j = 1,..., J;1. Initialize  according to equation (6) given βm-1)  (by equation (9) given , , σm-1) (= σm) (according to equation (5), and set ;5. Calculate weight βm) (given σm-1) (and  using equation (7), and set ;6. Calculate 7. Go to step 2 and iteratively update the estimators of parameters and the weights between steps 2 and 6 until convergence.DW devised and implemented the procedure described in the paper, drafted and finalized the manuscript. JH helped with writing and revising the manuscript. JH and MBS supervised and provided support for this work. HX and LM conducted the experiment that generated the data set used in the example.Methods section in this appendix. Again the notations from the Methods section:We derive estimation equations for location parameters presented in the b(x) = ,..., bK(x))',λi = '.φi(xij) can be approximated by a linear combination of B-spline basis functions, i.e. b'(xij)λi, where bk(xij) is the kth B-spline basis function of xij. Let A = ', C = ', and defineσ, the first partial derivatives of S (3) with respect to λ and β can be expressed in the matrix form asGiven scale parameter iB; = (b(i1x), b(i2x),..., b(iJx))', i y= ', ψ(x) = ρ'(x). As defined in equation (5)where andWi = diag,Wi into equations (10) and (11) and setting them to zeros, and solving these two equations and yielding estimation equations for  in equation (6) and  in equation (7). They arePlugging Letthen equation (7) can also be expressed as = (Z'WZ)-1 Z'W(y - B).Z'WZ)-1 can be explicitly calculates using the following matrix,The solution of .for ψ and χ functions derived from Huber's ρ(z) function areThe The related weight function has the formwhere H is a constant.α used in the scale step for Huber's robust estimation can be calculated as the followingThe constant Φ is the distribution function of the standard normal distribution, N is the total number of observations in the dataset, and p is the total number of parameters in the model.where ψ and χ functions derived from Tukey's ρ(z) function areThe The associated weight function has the formk is a constant and the constant a in the scale step takes valuewhere  is the Chi-square probability function with n degrees of freedom evaluated at s.where σ instead of iteratively updating equation (9) in our R program. It can be shown that equation (8) for Tukey's χ(z) function has an unique real root. This real root is just the solution for the estimator of σ. Let n* be the total number of observations that satisfy the second case of Tukey's χ function, i.e. | z | >k, let J* be the total number of clones that satisfy the first case of the χ, i.e. | z | ≤ k. Replacing z by  and plugging Tukey's χ into equation (8), we getWhen Tukey's weight function is used, equation (8) is solved directly for the estimator of whereLetThen equation (12) becomesax3 + bx2 + cx + d = 0.     (13)x = y - b/3a, divided by a in the both sides of equation (13), and plugs x into equation (13), then we get the Cardan's cubic equationLet p = c/a - b2/3a2, q = d/a - bc/(3a2) + 2b3/(27a3), the above equation becomesLet y3 + py + q = 0.     (15)The determinant for Cardan's equation (15) isq cannot be zero. If we can show that the p is greater or equal to zero, then the Δ must be positive. BecauseIt can be shown that the determined function Δ must be positive. The first term in the determinant equation must be positive because of the square function and ac - b2 is non-negative. We can see thatSo the Δ is positive if only if 3According to the Cauchy inequality , we havep must be non-negative and the Δ must be positive. Thus there is only one real root for equation (15), that isTherefore, the σ in equation (12) isThen the solution for