A new study settles a long-standing dispute about the genesis of an endangered species. With scant fossil evidence supporting a prehistoric presence, scientists could not say for sure where Borneo's elephants came from. Did they descend from ancient prototypes of the Pleistocene era or from modern relatives introduced just 300–500 years ago? That question, as Fernando et al. report in this issue, is no longer subject to debate.Applying DNA analysis and dating techniques to investigate the elephants' evolutionary path, researchers from the United States, India, and Malaysia, led by Don Melnick of the Center for Environmental Research and Conservation at Columbia, demonstrate that Borneo's elephants are not recent arrivals. They are genetically distinct from other Asian elephants and may have parted ways with their closest Asian cousins when Borneo separated from the mainland, effectively isolating the Borneo elephants some 300,000 years ago.In the 1950s, Borneo elephants had been classified as a subspecies of Asian elephants (either Indian or Sumatran) based on anatomical differences, such as smaller skull size and tusk variations. This classification was later changed, partly because of the popular view that these animals had descended from imported domesticated elephants. Until now, there was no solid evidence to refute this belief and no reason to prioritize the conservation of Borneo elephants.Their new status, as revealed by this study, has profound implications for the fate of Borneo's largest mammals. Wild Asian elephant populations are disappearing as expanding human development disrupts their migration routes, depletes their food sources, and destroys their habitat. Recognizing these elephants as native to Borneo makes their conservation a high priority and gives biologists important clues about how to manage them.


There is a much-quoted saying, attributed to the epidemiologist Geoffrey Rose: “A large number of people exposed to a small risk may generate many more cases than a small number exposed to a very high risk.” This is true for many individual risk factors such as salt intake (linked to high blood pressure and cardiovascular disease) and speeding on the highway (linked to injuries and accidents). Does it apply to many other global health risks? The study by Anthony Rodgers and colleagues suggests that it does.To develop effective health policies, one must understand the existing health risks and disease burdens. On a worldwide scale, this is a tough challenge. The Global Burden of Disease Database, maintained by the World Health Organization (WHO), collects data from countries around the world on risk factors such as tobacco, malnutrition, childhood abuse, unsafe sex, childbirth, and cholesterol levels, as well as on disease burdens, for example depression, blindness, and diarrhea. A large group of scientists from all over the world has developed a framework to analyze these data. To compare different risks or burdens, they calculate disability-adjusted life-years, or DALYs—the number of healthy life years lost because of a particular disease or risk factor.Rodgers and colleagues used data from the WHO database for 26 risk factors and from 14 epidemiological subregions of the world to calculate the proportion of risk-factor-attributable disease burden in different population subgroups defined by age, sex, and exposure level. For being underweight in childhood, for example—the leading risk factor for global loss of healthy life—they found that only 35% of the disease burden occurred in severely underweight children, the rest occurred in those only moderately underweight. The relative risks for the moderately underweight are much lower, but the number of children in that category is so large that the total attributable burden amounted to almost two-thirds of the total global burden of disease for that risk factor.The analysis confirms—and extends to a global level—previous research showing that many major health risks are important across the range of exposure levels, not just among individuals exposed to high levels of risk. It also points to risk factors that are particularly prevalent among specific populations and age groups, and for which highly targeted interventions could be effective.Despite numerous caveats and limitations of studies like this one, such analyses are essential aids in guiding the distribution of limited funds to lower the burden of life years lost to premature death and disability.


In this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR.vs. 0.052 ± 0.011 N, femoris 0.045 ± 0.005 N vs. 0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N vs. 0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue.Histochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA . In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N The results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process. One of the features characterizing mammalian skeletal muscle tissue is the structural variability of muscle fibres. The inclusive properties of skeletal muscle lead to functional diversity of muscle fibres, which has been related to differences in relative proportions of membrane structures and to different amounts of contractile proteins . Althoug2+-release via the ryanodine receptor  and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml-1 . Total p-1 . Myosin 2 (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution  at room temperature for 45 min. After incubations in 11 mM CaCl2, 2% CoCl2, and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl2, adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha , equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine.The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance . The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software.DHP; dihydropyridineDHPR; dihydropyridine receptorE-C coupling; excitation-contraction couplingFOG; fast oxidative glycolyticmusculus gastrocnemiusGAS; musculus gluteus maximusGLU; mATPase; myosin adenosine triphosphataseMHC; myosin heavy chainmusculus rectus femorisRF; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresisSR; sarcoplasmatic reticulumT-tubule; transverse tubuleIIa, IIb, IId; myosin heavy chain typeIIA, IIB, IID; fibre typeSM carried out most of the experiments, assisted in interpretation of results, and participated in writing the manuscript. MJ conceived of the study, participated in its design and coordination, and participated in writing the manuscript.


Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates.K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes.Ten clinical isolates, identified as E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.Clinical  Klebsiella pneumoniae isolates as the predominant ESBL-producers [Enterobacter aerogenes. It is also known that most of the E. aerogenes isolates in the Belgian hospitals belong to one of two predominant Belgian clones (BEI and BEII) [K. pneumoniae or K. terrigena by conventional biochemical testing were in fact E. aerogenes as could be shown by the use of genotypic methods, i.e. tDNA-PCR, validated by 16S rRNA gene sequencing, and by extensive phenotypic testing (including subculture and prolonged incubation).Enterobacteriaceae with β-lactam resistance due to the production of Extended-Spectrum β-Lactamases (ESBL) were discovered in the eighties and since that time became epidemic and endemic in hospitals worldwide . Since troducers  to predond BEII) , a situand BEII) . During E. aerogenes, K. pneumoniae or Raoultella (Klebsiella) planticola, the second code did not yield any identification, and the third code resulted in a weak but acceptable identification as K. terrigena. The isolates with the codes 5205773 and 5205753 were identified as K. pneumoniae by additional biochemical testing due to negative reactions for motility (tested in semi-solid agar) and ornithine decarboxylase. However, these isolates all possessed an inducible cefalosporinase, as detected on the antibiogram using a disk approximation test, a finding which strongly contradicts an identification as K. pneumoniae or Klebsiella sp.For ten clinical isolates, i.e. seven ESBL-producing clinical isolates collected during 2001 and three more recently collected isolates  and K. pneumoniae (BG) isolates, were tested in two different systems, i.e. Vitek 2  and Phoenix . Both automated systems yielded the same results as the API20E, i.e. that the E. aerogenes isolates, aberrant due to a slow reaction for motility and ornithine decarboxylase, were misidentified as K. pneumoniae  and antagonism (flattening of the inhibition zone), which is suggestive for a combination of an ESBL and an inducible β-lactamase. Again, inducible β-lactamases are very rare in Klebsiella spp. but typical for Enterobacter spp. It should be noticed that this phenomenon will not be detected by automated MIC-determination systems like Vitek 2 and Phoenix. Using PCR and sequencing as described previously [Disk diffusion antibiotic susceptibility testing, carried out according the NCCLS guidelines, revealed basically the same resistotype for all isolates, characterized by resistance to ceftazidime and susceptibility to ceftriaxone. Additional resistance to aztreonam was observed for some isolates, reflecting the most dominant resistance patterns for the E. aerogenes.The genotypic relationship of the phenotypically aberrant isolates was investigated with AP-PCR. Isolates GA1, GA2, GA3, MN2, MN3 and DBH were corresponding to Belgian clone BEI . species  which sh species  confirm mobilis" , which iKlebsiella spp., and an identification as Klebsiella sp. should warrant further investigation.However, the combination of an inducible β-lactamase and/or high-level cefoxitin resistance, which are rare in E. aerogenes misidentified as K. pneumoniae or K. terrigena due to delayed or negative ornithine decarboxylase and motility was reported previously, and was also discovered because of unexpected imipenem resistance of the so-called K. pneumoniae isolates [The phenomenon of isolates . Also inE. aerogenes as K. pneumoniae (or even K. terrigena) is probably not uncommon and probably also geographically widespread. This can be deduced from the following considerations: i) this phenomenon of misidentification of E. aerogenes was already reported in 1993 [The problem of misidentification of  in 1993 , ii) theKlebsiella species and of Enterobacter aerogenes, but do not mention the problem of possible delayed activity, possibly also because the study started from validated strains of each species.On the other hand, the problem seems to be largely unknown. In a recent study, Hansen and colleagues  carried E. aerogenes and K. pneumoniae. Here we showed that E. aerogenes isolates exist for which ornithine decarboxylase and motility are negative or delayed positive, and that as such these isolates can be misidentified as K. pneumoniae. This phenomenon may be quite frequent and geographically widespread. Genotypic identification techniques like tDNA-PCR, which moreover are cheaper than phenotypic testing for many bacterial species, can be semi-automatized, are faster and mostly have a higher discriminatory power, which is also reflected in this study.Identification in a routine clinical microbiology laboratory of the most commonly encountered Enterobacteriaceae is usually considered to be fast and straightforward, but apparently identification problems may occur due to diminished or delayed expression of some characteristics, even for well-established species like tDNA-PCR was carried out using the outwardly directed tRNA-gene consensus primers T5A (5'AGTCCGGTGCTCTAACCAACTGAG) and T3B (5'AGGTCGCGGGTTCGAATCC), thus amplifying the intergenic tRNA-spacers, as described previously ,11. Elec).For five of the phenotypically aberrant isolates, the complete 16S rRNA sequence was determined by amplification of the 16S rRNA-gene with the primers 5'-AGTTTGATCCTGGCTCAG and 5'-TACCTTGTTACGACTTCGTCCCA , and seqThe genotypic relationship of the isolates was investigated using arbitrarily primed PCR with RAPD Ready-to-Go beads  and the ERIC II primer 5'-AAGTAAGTGACTGGGGTGAGCG . AnalysiGC, GV and AM were responsible for sample collection and initial biochemical identification. PVD and GW carried out automated biochemical identification. GW in addition carried out extended biochemical characterization. TDB and MV carried out the molecular analysis. GC, GV, TDB and MV drafted the manuscript. All authors read and approved the final manuscript.


Over recent years increased emphasis has been given to performance monitoring of NHS hospitals, including overall number of hospital readmissions, which however are often sub-optimally adjusted for case-mix. We therefore conducted a study to examine the effect of various patient and disease factors on the risk of emergency medical readmission.The study setting was a District General Hospital in Greater Manchester and the study period was 4.5-years. All index emergency medical admission during the study period leading to a live discharge were included in the study . A multivariable proportional hazards modelling was used, based on Hospital Episodes Statistics data, to examine the influence of various baseline factors on readmission risk. Deprivation status was measured with the Townsend deprivation index score. Hazard ratios (HR) and associated 95% confidence intervals (CI) of unplanned emergency medical admission by sex, age group, admission method, diagnostic group, number of coded co-morbidities, length of stay and patient's deprivation status quartile, were calculated.Significant independent predictors of readmission risk at 12 months were male sex , age , number of coded co-morbidities (HR for >4 coded co-morbidities: 1.49 CI: 1.26–1.76), admission via GP referral  and primary diagnosis of heart failure  and chronic obstructive pulmonary disease/asthma . Higher level of deprivation was also significantly and independently associated and with increased emergency medical readmission risk at three , six  and twelve months .There is a potential for improving health and reducing demand for emergency medical admissions with more effective management of patients with heart failure and chronic obstructive airways disease/asthma. There is also a potential for improving health and reducing demand if reasons for increased readmission risk in more deprived patients are understood. The potential influence of deprivation status on readmission risk should be acknowledged, and NHS performance indicators adjustment for deprivation case-mix would be prudent. The 200Unplanned hospital re-admissions may represent adverse events and could therefore indicate poor quality of care,4. The iAlthough current NHS performance indicators use readmission rates at 28-days,2, thereIt can be hypothesised that factors such as patient sex, length of stay, number of coded co-morbidities, method of admission , as well as patient deprivation, independently influence the probability of hospital readmission. Lower socioeconomic status in particular is independently associated with increased risk for many adverse health outcomes, includiThis work was carried out as part of the routine function of Stockport NHS Trust Clinical Effectiveness Unit, and in relation to work commissioned by the then "Emergency Demand Management Group" of the Stockport Primary Care Trust. The objective was to accurately describe the epidemiology of emergency medical readmissions so that demand management strategies  could be informed.Stockport NHS Trust is a district general hospital in Greater Manchester, serving a reference population of about 300,000. About 85% of all patients emergency medical admissions are from Stockport, a population with a slightly lower, compared to the England and Wales average, Standardised Mortality Ratio from all causes  of 96 (95% CI 94–98).Hospital Episodes Statistics Data from April 1997 to September 2001 for Stockport NHS Trust were analysed and all emergency medical admissions in Stockport residents leading to live discharge were identified. An emergency medical admission was defined as an emergency hospital admission to any medical specialty in person over 18 years of age. Some persons had more than one emergency medical admission during the study period, and emergency medical admissions other than the index admission  were excluded. This was because not restricting analysis to index admissions would have meant that any deprivation gradients in readmission rates would have been confounded by deprivation gradients in index admissions, as previously described. This isAn emergency medical re-admission was defined as the first subsequent emergency medical admission during a follow-up period of either 28-days, or 3, 6 and 12 months respectively, following a first (index) emergency medical admission that led to a live discharge, and through the use of a single patient identifier. Observations were censored at the end of the chosen follow-up periods (as above) or at the time of intervening death unrelated to readmission to the study hospital. The latter was ascertained by data-linking to the Stockport Health Authority Public Health Mortality File produced by the Office for National Statistics.Index admission data were originally available on: sex; age; length of stay of index admission; International Classification of Diseases (ICD)-10 coded primary diagnosis; number (up to four) of coded co-morbidities; patient post code and admission method . Information on primary diagnosis was aggregated into five categorical groups comprising chronic obstructive pulmonary disease / asthma (ICD codes J44.0–45.9 respectively), heart failure (I50.0–50.9), acute coronary syndrome (I20.0 [unstable angina] and I21.0–9 [acute MI]), stroke (I60.0–I67.0) and all other conditions . Length of stay was divided into quartiles . Deprivation status was subsequently ascribed with an area-based measure, using the 1991 Census enumeration district (ED) of patient's post-code, and by the use of Townsend multiple deprivation index score. Four deprivation groups were defined, using quartiles of the range of the Townsend scores between Stockport EDs.sex, age group, diagnostic group (defined as above), admission method, number of coded co-morbidities (0–4), length of stay group (quartile) and deprivation group (quartile). Statistical significance for each of the above variables was assessed by the log rank test. A series of proportional hazards models with follow-up at 28 days, 3, 6 and 12 months were subsequently constructed to examine the adjusted hazard (risk) ratio of emergency medical readmission. Each model included all variables found to be significant in the uni-variable analysis at the 0.05 probability level. The proportional hazards assumption was tested using the Schoenfeld residuals as per the stphtest command in STATA. This showed that a time varying co-variate should be included in all four models, in relation to the length of stay variable (i.e. that an interaction term between length of stay and time of follow-up should be included), and this was hence included in the models.Kaplan-Meier readmission-free curves at 28 days, and 3, 6 and 12 months were constructed for each of the following variables: Additionally, for the number of coded co-morbidities and deprivation group variables, a test for trend was performed, entering the actual values as continuous variables. In this context, the test for trend value indicates the proportional change in the risk of readmission associated with one unit change in the exposure variable .There were 21,118 index emergency medical admissions corresponding to an equal number of patients leading to a live discharge during the study period, but primary diagnosis information was only available for 20,209 index emergency medical admissions . Test for trend confirmed the strong and statistically significant effect of deprivation at 3–12 months but there was no effect at 28 days.Admission method via a GP referral was significantly associated with a lower readmission risk at one year, but not at any other time intervals.Length of stay of the index admission influences readmission risk differently, depending on the length of follow-up as there was a highly significant interaction between length of stay group and time of follow-up see . Taking The findings indicate that in the study hospital about a quarter of patients with an index emergency medical admission will be readmitted in the same hospital during the subsequent year. Male sex and older age were strongly and independently associated with higher readmission risk, along with diagnosis of heart failure and chronic obstructive pulmonary disease or asthma. Effective measures to reduce readmission rates for patients suffering from these two conditions in particular are available,14 but nAs originally hypothesised, patient deprivation status exerted a significant independent effect on the risk of emergency medical readmission at 3–12 months of follow-up, with more deprived patients having had a higher readmission risk. There are several theoretical reasons why deprived patients may be at higher readmission risk, including: disease factors, such as greater disease severity in deprived patients; patientAlthough this is a single-centre study, the results may also have implications for the way current and future NHS performance indicators relating to readmission rates are both constructed and interpreted. NHS hospitals serving pre-dominantly deprived populations might in principle be disadvantaged if indicators are not adjusted for the impact of deprivation on case-mix. Although this study showed no significant effect of deprivation status on readmission risk at 28-days, which is the follow-up period currently used by the performance indicators,2, care The Department of Health includes performance indicators in the calculations of award of "three-star" status, which in turn is the "gateway" to "Foundation" status". StandarAll studies using administrative data are sensitive to the quality of routine data collection. The validity of HES data in relation to age, sex, length of stay, admission method, and area of residence is generally good, but misclassification errors may occur in relation to diagnostic codes and the extent to which co-morbidities are recorded and coded. CurrentA limitation of the study is that, besides the very large sample size, the findings are based on one single hospital in an urban English setting, and in principle the results are not generalisable. Similarly, the study was not population-based, so readmissions that may have occurred to other hospitals  were not ascertained. In theory such readmissions may have occurred at a differential rate between different deprivation groups. However this factor is unlikely to have biased the results in any considerable way for two reasons. First, the emergency (as opposed to elective) nature of the studied condition  makes it unlikely that either patients or doctors exercise an important degree of choice on which hospital a patient is admitted or readmitted, independently of patient deprivation status. Second, due to local geography and service configuration, 85% of the total medical admissions in Stockport residents occur at Stockport NHS (unpublished data). Similarly, by the nature of the hospital-centred nature of the study, admissions to private hospitals could not have been accounted. However, most admissions to private hospitals are for elective surgical procedures  for which we believe this is unlikely to have introduced considerable degree of bias. Lastly it is worth remembering that current NHS performance indicators for hospital Trusts are not population-based.Our study suggests that there is an important potential for both managing emergency demand and improving individual patient experience by focusing on the effective management of heart failure and chronic obstructive pulmonary disease. Although there is a similar potential by reducing differentials in readmission risk between deprivation groups, more research is required in order to understand reasons for such differentials in order to inform relevant policy measures. In the mean time, standardisation or other adjustment of hospital readmission indicators for patient socio-economic status in the future would be prudent. Failure to do so may disadvantage hospitals serving primarily deprived communities.The author(s) declare that they have no competing interests.The study was conceived and designed by GL and GC. GL, DH and IG analysed data. All authors contributed in the interpretation of findings and in the writing of the paper. GL and GC are guarantors.The pre-publication history for this paper can be accessed here:Length of stay group and readmission free Kaplan-Meier curves (0–28 days). This file demonstrates that during follow-up of 0–28 days, length of stay is not proportional to readmission risk, reason for which a time varying co-variate was included in the Cox regression model (see main article Text).Click here for file


Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression. Caliciviridae is made up of four genera, Sapovirus, Norovirus, Lagovirus, and Vesivirus, which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively. Human SaV and NoV strains are agents of gastroenteritis. The prototype strain of human SaV, the Sapporo virus, was originally discovered from an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, in 1977 [The family  in 1977 . The SaVCaliciviridae.Human SaV and NoV strains are noncultivable, but expression of the recombinant VP1 (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV ,4 In a rThe purpose of this study was to compare the time-course expression of two different SaV rVP1 constructs in a baculovirus expression system by Northern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), and EM. Our novel results have indicated that nucleotide point mutations increased the yields of SaV VLPs in insect cells, offering an alternative explanation for the increased expression levels of rVP1 and yield of VLPs.6 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. RNA transcription and rVP1 expression experiments were run in parallel for the Wt and MEG-1076 constructs.Expression of SaV rVP1 in a baculovirus expression system results in the self-assembly of VLPs . HoweverTotal RNA was extracted from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 days postinfection (dpi) for Wt and MEG-1076 constructs. Equal amounts (500 ng) of total RNA were added to a 2% agarose gel containing formaldehyde and stained with SYBR Gold Fig. . The rVPWestern blot analysis was used to compare the expression levels of Wt and MEG-1076 rVP1. The culture medium was separated from the cell lysate 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the Materials and Methods. Equal volumes of culture medium and cell lysate at each dpi were used for both constructs. Proteins were separated by SDS-PAGE, electrotransferred to PVDF, and detected with a 1:3000 dilution of hyperimmune rabbit Mc114 VLP antiserum. A band at the predicted rVP1 size (60 K) was first detected in the culture medium at 2 and 4 dpi for MEG-1076 and Wt constructs, respectively, which increased each day thereafter as evidenced by an increase in band intensity Fig. . As indiA thin band of approximately 55 K was also detected in the culture medium that appeared at 4 and 5 dpi for Wt and MEG-1076 constructs, respectively, and increased each day thereafter. In a different experiment, we determined the amino acid sequence of the MQG-1076 upper and lower bands by an Edman's degradation method. We discovered that the first three amino acid residues were MQG for both the upper and lower bands. This result indicated that the 55 K bands for these constructs were likely truncated or C-terminal deleted forms of rVP1. A thin band of 60 K was detected at every dpi in the cell lysate for the MEG-1076 construct Fig. , howeverE. coli expressed VP2 (data not shown).The VP2 amino acid sequence was the same in all constructs. We did not detect rVP2 during the time-course expression of the MQG-1076 construct using the antiserum raised against An antigen ELISA system was used to compare the yields of Wt and MEG-1076 VLPs at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The ELISA incorporated hyperimmune rabbit (capture) and guinea pig (detector) antisera raised against purified Mc114 VLPs [The MEG-1076 construct contained a nucleotide point mutation in which resulted in an amino acid substitution at position 358 in VP1. We aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found this residue was strictly conserved, but more importantly, there was a strictly conserved amino acid motif at this site, NGDV (data not shown). However, when we included a porcine SaV GIII strain and a recently identified SaV GIV strain , only the GD site was strictly conserved, though several other amino acids nearby were also strictly conserved  had decreased levels of rVP1 mRNA at 36 hours post-infection, by approximately 50%, when compared to a construct without the mutation (ORF2+ORF3+3' UTR construct). Bertolotti-Ciarlet suggested that the RNA secondary structure or changes in the mRNA stability could be responsible for the different steady-state levels, but this was not proven.In this study, we compared the time-course expression of two different Mc114 SaV rVP1 constructs in a baculovirus expression system . Another explanation may have been differences in the extents to which these baculoviruses induce apoptosis and all these may result from features in the baculovirus skeleton rather than from the inserted SaV sequence. Such effects might for instance affect the number of adherent cells harvested or the degradation rates of both proteins and RNAs. However, we found that the MQG-1076 construct, developed from a separate experiment, had similar expression levels to that of the MEG-1076 construct (data not shown), which may eliminate the possibility that the baculovirus skeleton played a role in the increased yields of VLPs. On the other hand, we could not demonstrate whether the nucleotide mutations in VP1 and/or in ORF2 affected the transcription, a construct with only one of these mutations would be needed. Nevertheless, our results indicate that translation was exclusively affected by the single amino acid substitution in VP1. Therefore, the final increase in yields of VLPs may have been coupled at multiple levels, involving one or both of the nucleotide mutations in VP1 and VP2.We did not detect rVP2 during the time-course expression of the MQG-1076 construct (data not shown). The Wt and MEG-1076 constructs had an identical amino acid sequence, which would suggest a similar negative-result. NoV studies have found that inclusion of VP2 increases the stability of VLPs, though the expression level of NoV rVP2 was low [The amino acid substitution (N → S) for the MEG-1076 construct occurred in the VP1 gene at residue 358. This asparagine residue was recently identified as a moderately conserved residue among the caliciviruses capsid proteins [Caliciviridae and identified strictly and moderately conserved residues, and predicted the P1 and P2 domains of SaV VP1 based on NoV X-ray crystallography structure. Based on these predictions, the residue at position 358 (amino acid sequence) was found as a moderately conserved residue among the caliciviruses. This arginine residue was predicated to be in the P2 domain, which is defined as the outer most protruding domain for NoV and thought to provide strain diversity [Recently, the cryo-EM analysis of SaV was determined and compared to NoV X-ray crystallography structure . Chen etiversity . FurtherSaV GI Mc114 strain  was isolated from a male infant seven months of age from the McCormic Hospital, Chiang Mai, Thailand on the 7th May 2001 . RNA extGAG → CAG) and 1076 (AAT → AGT) and one nucleotide point mutation in ORF2 at position 1895 (GTG → GTA) (relative to the VP1 start and represented in bold). Primer and PCR errors likely introduced these mutations. These three nucleotide point mutations resulted in two amino acid substitutions in the VP1 gene, one at the second residue, where glutamic acid (E) → glutamine (Q), and one at residue 358, where asparagine (N) → serine (S). The nucleotide point mutation in ORF2 did not result in an amino substitution. Despite the two amino acid substitutions, the MQG-1076 construct formed VLPs. We designed another construct (MEG-1076) using the pDEST8-MQG-1076 as template but with a new sense primer and used KOD-plus DNA polymerase according to the manufacture's instructions . The MEG-1076 construct had the same nucleotide point mutations at positions 1076 in VP1 and 1895 in VP2 as the MQG-1076 construct but not at nucleotide 4 in VP1  was amplified with ExTaq DNA polymerase. However, this construct was later found to have two nucleotide point mutations in ORF1 at positions 4 , and then resuspended in 30 μl of Grace's medium. The cell lysate from the first centrifuge was resuspended in 200 μl of Grace's medium and stored at 4°C.Recombinant bacmids were transfected into Sf9 cells  and the recombinant baculoviruses was collected as previously described . The expg, the supernatant removed, and then the cells were dissolved with 1 ml of Isogen. The cells were stored at -80°C. RNA was purified by a chloroform/ ethanol method . Briefly, RNA was mixed with chloroform, centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous layer collected. This was repeated once, and then the aqueous layer collected and mixed with isopropanol and stored overnight at -20°C. The solution was mixed, centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant discarded. The pellet was resuspended in 80% ethanol, centrifuged at 12,000 × g for 15 min at 4°C. This was repeated once, and then the pellet air-dried and resuspended in 25 μl of TE, and stored at -80°C. The amounts of purified RNA were determined spectrophotometrically . The same amounts (500 ng) of total RNA were loaded for each construct and each dpi onto a 2% denaturing agarose gel containing formaldehyde. The amounts of total RNA were compared using SYBR Gold staining . RNA was transferred to a positively charged nylon transfer membrane  under vacuum  and analyzed by Northern blotting according to the DIG Northern Starter Kit , except for a minor modification. Briefly, a RNA probe corresponding to Mc114 VP1 position 157 to 1283 (anti-VP1) was generated from a PCR fragment (native sequence) according to the manufacture's instructions . Hybridization was performed overnight at 68°C with anti-VP1 in 10 ml of ultrasensitive hybridization buffer . After hybridization, immunological detection was performed according to the manufacture's instructions .Total RNA was prepared from the attached cells at 1, 2, 3, 4, 5, and 6 dpi with 1 ml of Isogen . For 7 and 8 dpi, the cell culture medium (containing unattached cells) was collected and centrifuged for 5 min at 3,000 × Western blotting, ELISA, and EM were used to examine rVP1 expression as previously described . However. In total, we aligned different 21 SaV GI, GII, GIII, GIV, and GV sequences, and included: Arg39, AY289803; Bristol, AJ249939; C12, AY603425; Cruise ship/00, AY289804; PEC, AF182760; Dresden, AY694184; Hou-7, AF435814; Houston/86/US, U95643; Houston/27/90/US, U95644; London/29845/92/UK, U95645; Lyon/598/97/F, AJ271056; Manchester, X86560; Mc2, AY237419; Mc10, AY237420; Mex340/1990, AF435812; Mex14917/00, AF435813; NK24, AY646856; Parkville, U73124; Potsdam, AAG01042; Plymouth, X86559; Sapporo/82/Japan, U65427; and Sakaeo-15, AY646855.VP1 nucleotide sequences were translated using Genetyx software (software development Co. Version 11.2.2) and submitted to online ClustalW at DDBJ The author(s) declare that they have no competing interests.GH carried out the study and wrote the manuscript. KK, TO, KN, and NT participated in the design of the study and helped to draft the manuscript.


In the hypothesis that the increased collagen metabolism in the intestinal wall of patients affected by inflammatory bowel disease (IBD) is reflected in the systemic circulation, we aimed the study to evaluate serum level of procollagen III peptide (PIIIP) in peripheral and splanchnic circulation by a commercial radioimmunoassay of patients with different histories of disease.Twenty-seven patients, 17 with Crohn and 10 with ulcerative colitis submitted to surgery were studied. Blood samples were obtained before surgery from a peripheral vein and during surgery from the mesenteric vein draining the affected intestinal segment. Fifteen healthy age and sex matched subjects were studied to determine normal range for peripheral PIIIP.In IBD patients peripheral PIIIP level was significantly higher if compared with controls ; splanchnic PIIIP level was 5.5 ± 2.6 μg/l showing a positive gradient between splanchnic and peripheral concentrations of PIIIP. No significant differences between groups nor correlations with patients' age and duration of disease were found.We provide evidence that the increased local collagen metabolism in active IBD is reflected also in the systemic circulation irrespective of the history of the disease, suggesting that PIIIP should be considered more appropiately as a marker of the activity phases of IBD. Crohn's disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of unknown origin of adolescent and young adulthood  where geCurrent opinions suggest that an increased synthesis of collagen type I, III, and V may play an important role in the pathophysiological mechanism leading to intestinal fibrosis -15. An iHowever, not all authors are in agreement regarding the increased serum levels of the aminoterminal propeptide (PIIIP) of collagen in peripheral and splanchnic circulation of patients with active IBD ,14. BeloTwenty-seven patients affected by active IBD, 17 with CD , and 10 with UC  submitted to surgery, were enrolled in the study in a double blind fashion. The protocol was approved by local Ethical Committee and informed consent was obtained from all participants to the study. Three patients had CD in small bowel only, two in large bowel only, 12 had ileocolonic disease. Disease activity was assessed according to the Crohn's Disease Activity Index (CDAI)  and the Patients affected by CD were operated on for recurrent obstruction, whereas patients with UC were submitted to surgery because of refractory to medical therapy.The clinical diagnosis was confirmed by histology Fig. ; all cas- for CD: deep ulcers, marked proliferation of small lymphoid nodules involving all layers of intestinal wall sometime with sarcoid-type granulomas and serosal inflammation;- for UC: mucosal erosions and superficial ulcerations usually limited to the upper submucosa with cryptic abscesses and glandular destruction.The clinical profile of the studied patients is reported Table . Two patDifferent kinds of collagen have been identified in humans. All of them derive from longer precursor molecules (procollagens). They are synthesized intracellularly and secreted in extracellular space where they are cleaved by aminoproteases -23. AmonData were analyzed using a computer statistical software . All the quantitative variables were tested for Gaussian distribution with the Kolmogorov-Smirnov test. All that followed this distribution were presented as mean ± standard deviation.Differences at baseline in collagen parameters between IBD patients and controls were tested for significance using the analysis of variance with the Bonferroni correction. The relation between collagen parameters and the estimated duration of the disease and indices of disease were tested with regression analysis. In all cases, a p value less than 0.05 was considered significant.At baseline, before surgery, serum p-PIIIP in IBD patients were significantly higher if compared with healthy controls  fig . No signDuring surgery, serum s-PIIIP in IBD patients was 5.5 ± 2.6 μg/l. No significant differences were found when comparing CD and UC subgroups . A positive gradient was found in IBD patients between splanchnic and peripheral serum concentrations of PIIIP (0.7 ± 1.9 μg/l). This gradient was confirmed when separately considering each disease, without significant differences between the two subgroups  fig .No significant correlation was found between peripheral and splanchnic levels of PIIIP and the age of the patients and the estimated duration of the disease. Regarding the activity indices, the number of patients belonging to each class was not enough to perform a statistical analysis. Notwithstanding, for the TWI in UC patients a significant difference in PIIIP levels was found between mild and severe form of the disease Table . FinallyCD and UC are chronic pathologies characterized by an early onset followed by sporadic episodes of acute symptoms during lifetime, debilitating the affected patients to perform their daily functions . Until nIn the present study we have found that intestinal collagen metabolism in IBD patients was increased and that it is reflected in local and systemic circulation. Differently from some experiences ,13, we hWhen comparing the mild with the severe form of the disease, a significant difference in PIIIP levels was found only in patients affected by UC. This data will probably be confirmed when the number of patients enrolled in each disease-related activity categories is extended as presently in our series the majority of the patients were classified as severe.The effect of glucocorticoids on collagen synthesis, collagenase, and collagen degradation has not yet fully been clarified . In our In conclusion we provide evidence that collagen metabolism in IBD is reflected in the systemic and local circulation, without any differences between UC and CD, irrespective of the age of the patients and the duration of the disease. Therefore, this marker may give further information on the activity phases rather than on the entire history of the disease. Further data on the possible use of PIIIP as useful marker of choice for surgical option are attended from the follow-up at 6 and 12 months, which is still on-going .Inflammatory bowel diseases = IBDProcollagen III propeptide = PIIIPPeripheral Procollagen III propeptide = p-PIIIPSplanchnic Procollagen III propeptide = s-PIIIPCrohn's disease = CDUlcerative Colitis = UCCrohn's Disease Activity Index = CDAITruelove-Witts index = TWIThe author(s) declare that they have no competing interests.UC conception and design, interpretation of data, drafting the articleMDS conception and design, interpretation of data, drafting the articleECA performed surgical operations, critical revision of the article, final approval of the versionBO patients' enrollement, blood samples collectionRP statistical analysis, interpretation of data, drafting the articleAP echocardiographic studiesGB radioimmunoassaysEO radioimmunoassaysSF histological examinationsFM interpretation of data, critical revision of the article, final approval of the versionMMC conception and design, interpretation of data, drafting the articleAll Authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Balanced decisions about health care interventions require reliable evidence on harms as well as benefits. Most systematic reviews focus on efficacy and randomised trials, for which the methodology is well established. Methods to systematically review harmful effects are less well developed and there are few sources of guidance for researchers. We present our own recent experience of conducting systematic reviews of harmful effects and make suggestions for future practice and further research.We described and compared the methods used in three systematic reviews. Our evaluation focused on the review question, study designs and quality assessment.One review question focused on providing information on specific harmful effects to furnish an economic model, the other two addressed much broader questions. All three reviews included randomised and observational data, although each defined the inclusion criteria differently. Standard methods were used to assess study quality. Various practical problems were encountered in applying the study design inclusion criteria and assessing quality, mainly because of poor study design, inadequate reporting and the limitations of existing tools. All three reviews generated a large volume of work that did not yield much useful information for health care decision makers. The key areas for improvement we identified were focusing the review question and developing methods for quality assessment of studies of harmful effects.Systematic reviews of harmful effects are more likely to yield information pertinent to clinical decision-making if they address a focused question. This will enable clear decisions to be made about the type of research to include in the review. The methodology for assessing the quality of harmful effects data in systematic reviews requires further development. Systematic reviews are important tools for evidence-based health care. They are certainly one of the reasons for the progress that has been made in obtaining reliable evidence on the beneficial effects of interventions. A recent study of the medical literature, using Medline and the Cochrane Library, showed that the number of systematic reviews published has increased dramatically, from a single publication in the years 1966 to 1970, to 23 in 1981 to 1985, and 2467 in 1996 to 2000 [The methodology for conducting systematic reviews of beneficial effects from RCTs is well established, whereas the methods for systematically reviewing randomised or observational data on harmful effects are less well developed and less often used. Only 1.25% of 3604 publications cited in the 2001 edition of Side Effects of Drugs (SEDA-24) were systematic reviews [It is not uncommon, even among experienced reviewers, to assume that the objective of a systematic review of harmful effects should encompass all known and previously unrecognised harmful effects and that data from all types of study design should be sought. We have re-visited three systematic reviews of drug interventions in which we had reviewed harmful effects, to evaluate our own recent experience, identify areas for improvement and to share our ideas with other researchers undertaking reviews.We used three reviews for this study on the basis that they had been completed recently (between 2001 and 2003) and that one of us had been the lead reviewer of harmful effects in each review. The reviews were conducted as Health Technology Assessments for the National Coordinating Centre for Health Technology Assessment (NCCHTA) on behalf of the National Institute for Clinical Excellence (NICE). The reviews, in order of completion, were: nicotine replacement therapy (NRT) and bupropion sustained release (SR) for aiding smoking cessation , atypicaWe described and compared the methods used in each review and the problems we encountered in applying those methods. We focused our evaluation on the review objectives, the inclusion criteria for study design and the quality assessment of the primary studies. We do not report on the matter of searching for studies about harmful effects which presents another challenge to those who conduct systematic reviews ,11, becaThe main components of the three systematic reviews of harmful effects are described in Table The schizophrenia review objective appeared to be appropriate in seeking to determine the incidence of named outcomes that were considered by health economists to be most likely to lead to a change in prescribed treatment . The objAll three reviews included study designs other than RCTs to assess harmful effects. The types of non-randomised studies included for each review reflected differences in the reviews' objectives, our judgment as reviewers as to where the most useful data were likely to be found, and was to some extent pragmatic in terms of the time available to complete the reviews. The reviews with the broad objectives included more non-randomised studies and more diverse study designs. The schizophrenia and epilepsy reviews specified a minimum size and duration of study to be included (see table) in an attempt to add data over and above what was available from the largest and longest RCTs. Doing this did involve some indeterminable risk of missing important information.The review of observational studies carried out in the schizophrenia review was necessary because the pre-determined harmful effects of interest were known to be under-reported in RCTs . The incOnce the inclusion criteria for study design had been defined, applying them was problematic. Reports of primary studies rarely described the study design in sufficient detail. Many of the studies included in the schizophrenia review purported to be cohort studies but on closer examination were in fact large case series involving more than one intervention. Some of the 'cohort study' data on bupropion SR included in the smoking cessation review had actually been derived retrospectively from RCTs. How exactly the 'cohorts' had been established in studies of epilepsy was often unclear in terms of the source population, eligibility criteria, and selection, or was simply not reported. Had we, in all three reviews, only included reports of studies fitting textbook definitions of particular study designs, virtually all of the primary study reports we identified would have been excluded. The inclusive approach we took turned out to be unrewarding.In the smoking cessation review, in addition to difficulties with the study design inclusion criteria, application of the criterion to only include studies in which assessment of adverse events was the primary objective was problematic because it involved a high degree of subjective judgment.We encountered problems when applying published checklists in our reviews of harmful effects. The response to some questions depended on the outcome of interest, for example, follow-up may have been adequate for the assessment of the primary  outcome of the study but not for the collection of data on harmful effects. We also found that published checklists omit key features such as how harmful effects data were recorded. In the epilepsy review we were in a position to learn from the earlier reviews and spent time clarifying the questions in the checklists so that they would provide information relevant to the reliability of the harmful effects data. We also added items pertinent to reports of harmful effects such as how and when events were recorded and whether the time at which they occurred during the study was reported. Although this informed approach was a step in the right direction, the major hindrance to applying checklists in all three reviews was inadequate reporting of the basic design features of the primary studies.Once the quality criteria had been applied there remained the challenge of interpreting the results. In our reviews we described the evidence identified and tabulated the response to each checklist question for each primary study. This generated lengthy summaries that had limited utility. Even comparing validity within study designs (not across them) we found it impossible to synthesise the information as all the included studies had methodological flaws and features that could not be assessed due to inadequate reporting. Reaching a decision about which studies were likely to give the most reliable results was not straightforward.Our experience of reviewing harmful effects mirrors that of other researchers in that a significant investment of effort failed to yield significant new information ,17.A focused review question is standard practice for assessing beneficial outcomes in systematic reviews and should also be so when reviewing harms. Researchers conducting reviews need to make sure that they address a well-formulated question about harms that are likely to impact on clinical decisions. Focusing a review question about harmful effects will not necessarily mean restricting it to specific adverse events but may mean, for example, addressing a particular issue such as long-term effects, drug interactions, or the incidence of mild effects of importance to patients. If the aim of the research is to look for previously unrecognised harmful effects, analysis of primary surveillance data may be more appropriate than a systematic review . ResearcIt is important to realise that an unquestioning belief that observational studies are the best source of harmful effects data simply because they are not RCTs can be a pitfall. It is essential to think carefully about the review question before widening the inclusion criteria to include non-randomised study designs. Some harmful effects, such as very rare events or those emerging in the long-term, are unlikely to be addressed adequately in RCTs. But, even if observational studies are appropriate to the review question researchers should be prepared for the difficulty of interpreting observational study data outweighing the anticipated benefits.The importance of quality assessment of RCTs in systematic reviews of effectiveness is well established , but deba priori what exactly they need to glean from their quality assessment of the primary studies in their own review of harmful effects and it may be necessary to differentiate clearly between internal and external validity.It is essential that quality assessment is able to discriminate poor from better quality studies of harmful effects. Levels of evidence hierarchies have several shortcomings. The hierarchy of evidence is not always the same for all harmful or beneficial outcomes. For example, an RCT with adequate internal validity but limited sample size or follow-up may be a less reliable source of information about relatively uncommon harmful effects emerging in the long-term than a large well-conducted cohort study with many years of follow-up. Another problem with ranking evidence in a hierarchy is that different dimensions of quality get condensed into a single grade, resulting in a loss of information. Furthermore, the dimensions included in current hierarchies may not be the most important in terms of reflecting the reliability of a particular study's findings . ResearcWe suggest that further research is needed to collate, assimilate and build on the existing information relevant to systematically reviewing primary studies for harmful effects of health care interventions. This should include a review of the literature pertinent to the methodology of incorporating evidence of harmful effects in systematic reviews; a description and categorisation of the methods used in systematic reviews published to date, and any evidence from methodological research on which they are based; and the development of quality assessment methods.Appraisal of our recent experience highlighted some of the problems inherent in conducting systematic reviews of harmful effects of health care interventions. Such reviews need to address a well-formulated question to facilitate clear decisions about the type of research to include and how best to summarise it, and to avoid repeating what is already known. The review question about harmful effects needs to be relevant to clinical decision-making. A systematic review of the methodology pertinent to systematic reviews of harmful effects is warranted.None declared.AMB, HMM and NFW conducted the review work described. NFW conceived of the study. NFW and HMM drafted the manuscript. All authors contributed to, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Previous reports have indicated an excess of leukaemia in Broome County, New York, particularly in the Town of Union. Surveillance of cancer incidence data indicates that a large proportion of these cases occurred among males ages 65 and older. Shoe and boot manufacturing has been the largest single industry in this area throughout much of the past century. Occupational studies from Europe suggest a link between leukaemia and employment in the shoe and boot manufacturing industry. However, researchers have not found a positive association between leukaemia and employment in the shoe industry among workers in the United States.A matched case-control study was conducted to investigate the association between leukaemia incidence among males 65 and older and employment in the shoe and boot manufacturing industry. Thirty-six cases of leukaemia occurring between 1981–1990; among males age 65 and older; residing in the town of Union met the study case criteria. Death certificates were obtained for each of the cases. These were matched to death certificates of 144 controls on date of death and date of birth +/- 1 year. Death certificates were then examined to determine the employer and occupation of each study subject. Conditional logistic regression was used to determine the risk of leukaemia among those working in the industry.The risk of both leukaemia  and acute myeloid leukaemia  were elevated among those employed in the shoe and boot manufacturing industry, however neither was statistically significant.The results, though suggestive of an association between leukaemia and employment in the shoe and boot manufacturing industry, were not statistically conclusive due mainly to limited study power. Several additional limitations may also have prevented the observance of more conclusive findings. Better exposure assessment, information on length of exposure and types of job held, control of confounding factors and information on chemicals used by this company would strengthen any future investigation. A number of previous reports have indicated an excess of leukaemia in Broome County, New York, particularly in the Town of Union -5. The BSpatial analysis techniques have also been used to investigate clustering of leukaemia within central New York ,5. AnalyThe International Agency on Cancer Research (IARC) has classified employment in the boot and shoe manufacturing industry as a Group 1 risk factor, meaning there is sufficient evidence that the exposure or setting is carcinogenic to humans . A numbeA number of epidemiological reports have shown an association between employment in the shoe and boot manufacturing industry and an increased risk of leukaemia mortality among workers in Italy, Turkey and Great Britain -15. The The Endicott Johnson Company was the major employer in the Town of Union between 1930 and 1960 . The comThe objective of the current study is to investigate the association between leukaemia incidence and employment in the Endicott Johnson tanneries and shoe and boot manufacturing facilities, in the Town of Union, Broome County, New York.A matched case-control study was conducted to investigate the association between leukaemia incidence among males 65 and older and employment in the shoe and boot manufacturing industry. The New York State Cancer Registry served as the source for the leukaemia cases. Because the excess of leukaemia was limited primarily to males 65 and older, the selection of cases was restricted to males aged 65 and over with a primary diagnosis of leukaemia (ICD9 204–208) occurring between the years of 1981–1990 and residing in the Town of Union, Broome County, NY at the time of diagnosis. In addition, all cases must have been deceased as of August 1997 and a resident of the Town of Union at the time of death. Since acute myeloid leukaemia (AML) has been most closely associated with exposure to benzene, a subset of the original cases, which included only cases of AML (ICD9 205.0), was also examined. Once the cases were identified from the Cancer Registry, death certificates for each were obtained from the NYSDOH Vital Records Section.Death certificates for controls were obtained from the NYSDOH Vital Records Section. Four controls meeting the following criteria were selected at random for each case. Controls were restricted to males 65 years and older who resided in the Town of Union at the time of death. In addition, controls were matched to cases on year of death and year of birth +/- 1 year. Matching on dates of birth and death was done to control for potential confounding due to age and to ensure that the cases and controls had a similar opportunity of being exposed (i.e. to work at Endicott Johnson). Additionally, the controls must not have had leukaemia listed as either a cause of death or a contributing cause of death on their death certificates.Of the 41 leukaemia cases among males 65 and older listed in Table Occupation was determined from information on the death certificate because this represented the most readily available source of occupational information. No company or union records were easily available to determine employment or job status. The company has not allowed previous researches access to company records for occupational health studies . In addiFor the purpose of this study an individual was considered exposed if Endicott Johnson was listed on the death certificate under "Name and locality of firm or company". If this field was blank or the employer could not be determined, the fields "usual occupation" and "kind of business" were examined to see if the employer could be determined. The individual responsible for assigning exposure was blinded to case or control status of the study subjects. Because of the ambiguity of many of the listings of occupation on the death certificates, it was not possible to subdivide Endicott Johnson employees further by occupation in order to exclude those employees who had no exposure to carcinogenic chemicals or processes. Therefore all workers in the shoe and boot making factories as well as workers from the nearby Endicott Johnson tanneries were included among the group considered to be exposed.Crude odds ratios (OR) were first calculated for "exposed" versus "non-exposed" groups. However, since each case was individually matched to a set of four controls, the matched sets were analyzed as individual strata. An adjusted OR was therefore calculated taking into account just the contribution of the discordant pairs within each stratum . The SASApproximately 29% of the study subjects (cases and controls) were employed by Endicott Johnson. This was more than the next 5 largest employers in the area combined. Table Four study subjects (1 case and 3 controls) had shoe worker as their occupation but their employers were companies other than Endicott Johnson. Odds ratios and confidence intervals were calculated with the subjects considered both as exposed and unexposed. The results of the study were not significantly different. For the results presented here the four subjects were considered as not working for Endicott Johnson (i.e. unexposed). None of these study subjects were part of the AML subgroup.Overall those whose death certificates indicated that they worked at Endicott Johnson had approximately a 50% higher risk of developing leukaemia than those who did not work for the company; however, there was not enough evidence to rule out the possibility that the observed results were due to chance alone. Among the types of leukaemia, occupational exposures have been most closely associated with acute myeloid leukaemia; thus we might have expected to observe a greater risk of AML among Endicott Johnson workers. This was not the case, however, as the risk of AML was slightly less than that observed for all leukaemias.Previous studies of workers employed in the shoe and boot manufacturing industry in the United States have failed to find an elevated risk for leukaemia mortality -19. In cLimitations that may have prevented the observance of more conclusive findings including small sample size, limited work history, no exposure assessment and limited control of confounding factors. Table The power to detect a true increase in risk was limited by the population size of the study. Because this study was a follow-up to a previous study, the population was limited by the constraints of the previous study. Leukaemia is a relatively rare disease and the population of white males over 65 in the study area was only approximately 3,500. For the current study the power to detect an increased risk of 50% (similar to the observed risk for leukaemia) with a 95% confidence interval was only 20%. In order to achieve a power of 80% the study population would have had to have been approximately eight times as large. This could have been achieved by either increasing the study area to include neighbouring communities or by increasing the period under observation. However, as mentioned previously we were limited by the parameters of the previous study.The accuracy of occupation and industry data found on death certificates has been examined in a number of studies -27 and tIn addition, only dead individuals were eligible as controls in the study. Since working in the shoe and boot manufacturing industry is associated with other kinds of types of cancer, such as lung and bladder cancer, in addition to leukaemia, this may have artificially increased the prevalence among controls. This would also bias the odds ratio towards the null.A major limitation in this study was the assessment of exposure. Because death certificates were used to identify usual employer and occupation, information on specific jobs worked was fairly limited and often missing completely. Even when the employer was known to be Endicott Johnson, a nondescript job title such as "shoe worker", "Endicott Johnson worker", or "labourer" was often given for occupation. Because of this, any mention on the death certificate of having worked at Endicott Johnson was used as a surrogate for exposure. The effect of including every Endicott Johnson worker in the exposed group may have biased the odds ratio toward the null due to the large variety of jobs within each factory. In addition to all workers in the shoe and boot making factories, workers from the nearby Endicott Johnson tanneries were also included among the group considered to be exposed, further diluting the effect. Previous studies have found an increase in leukaemia mortality only among men in departments where shoes were assembled -15.Additionally, it was not known what chemicals were used in the production of shoes at the Endicott Johnson facilities. Therefore, it is difficult to know if the lack of an association between leukaemia and employment for Endicott Johnson may have been a result of poor exposure assessment or a result of no exposure to certain carcinogens.The length of employment and timing of the exposure were also not taken into account. However, the "usual occupation" as listed on the death certificate is most likely to represent long-term, stable employment. In Italy, the highest leukaemia rates were found among workers who worked in the industry prior to 1963 . EstimatMigration is a problem when investigating diseases such as cancer that have a long latency period. In the current study this may have been compounded by the fact that the workforce at Endicott Johnson has been declining since the mid 1950's as the company slowly scaled back and eventually eliminated its production facilities in the area. In addition, both cases and controls may have retired and moved out of the area. Overall, however, it is felt that those males age 65 and older in this region represent a fairly stable population. Nonetheless, any misclassification due to migration would certainly bias the results toward the null since it is unlikely that any cases who recently moved into the area would have worked for Endicott Johnson.Most types of bias identified in this study would tend to lead to an underestimation of the true risk (i.e. bias the results toward the null). A summary of the types of biases identified and their effects is given in Table In the study only age, gender and date of birth were controlled. Because of the study design, other possible confounders could not be taken into account. For instance, cigarette smoking has been associated with several forms of leukaemia, however no attempt was made to determine smoking status in the current study .The incidence of leukaemia was of interest in this community because a number of previous cancer investigations in the area have found an increase in leukaemia incidence. In addition, several studies have found an association between leukaemia and employment in the shoe and boot manufacturing industry, which was predominant in the town for most of the past century. In the current study, a positive association between the risk of leukaemia and working at Endicott Johnson was observed; however, the small population size prevented us from determining whether the results could be due to chance alone. Serious limitations may have prevented the observance of more conclusive findings in this study. Better exposure assessment, information on length of exposure and types of job held, control of confounding factors and information on chemicals used by this company would strengthen any future investigations. However, many of the most carcinogenic chemicals, which at one time were used in the industry, have not been used for several decades. In addition, since the company no longer manufactures shoes and boots, there is no current or future exposure among this particular group. Nonetheless, lessons learned from retrospective analyses of disease among workers in American industries may be applicable to overseas industries, particularly in developing nations, where many of the safeguards and restrictions that have been in place for decades in the US and Europe have not yet been adopted.AML, acute myeloid leukaemiaCI, confidence intervalIARC, International Agency on Cancer ResearchICD, International Classification of DiseasesNYSDOH, New York State Department of HealthOR, odds ratioNone declared.Not applicable.


This work presents a novel prokaryotic promoter prediction method based on DNA stability.In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Improved promoter prediction methods can be one step towards developing more reliable Escherichia coli, which have near 50 % G+C composition and also performs satisfactorily in case of other prokaryotic promoters.The promoter region is less stable and hence more prone to melting as compared to other genomic regions. Our analysis shows that a method of promoter prediction based on the differences in the stability of DNA sequences in the promoter and non-promoter region works much better compared to existing prokaryotic promoter prediction programs, which are based on sequence motif searches. At present the method works optimally for genomes such as that of Our analysis clearly shows that the change in stability of DNA seems to provide a much better clue than usual sequence motifs, such as Pribnow box and -35 sequence, for differentiating promoter region from non-promoter regions. To a certain extent, it is more general and is likely to be applicable across organisms. Hence incorporation of such features in addition to the signature motifs can greatly improve the presently available promoter prediction programs. One of the important steps towards ab initio gene prediction is to develop better promoter and TSS (transcription start site) prediction methods.Accumulation of a huge amount of genome sequence data in recent years and the task of extracting useful information from it, has given rise to many new challenges. One of the biggest challenges is the task of gene prediction and to fulfil this need, several gene prediction programs have been developed (For reviews see -5). MostAlthough reasonable progress has been achieved in the prediction of coding region, the promoter prediction methods are still far from being accurate -9 and thIn our earlier analysis, we observed that in case of bacteria as well as in eukaryotes, various properties of the region immediately upstream of TSS differ from that of downstream region . There aE. coli 0.49, B. subtilis 0.56 and C. glutamicum 0.46), show strikingly similar features. Promoters from all the three bacteria show low stability peak around the -10 region. The second prominent feature in the free energy profiles of all the three bacteria is the difference in stabilities of the upstream and downstream regions. In all the three groups of promoter sequences, the average stability of upstream region is lower than the average stability of downstream region. But the three sets of promoter sequences differ in their basal energy level, which seems to be dependent on the nucleotide composition of the bacteria.It is well known that the stability of a DNA fragment is a sequence dependent property and depends primarily on the sum of the interactions between the constituent dinucleotides. The overall stability for an oligonucleotide can thus be predicted from its sequence, if one knows the relative contribution of each nearest neighbour interaction in the DNA . The aveE. coli promoter sequences. Our statistical analysis using "Wilcoxon signed test for equality of medians" (see METHODS) shows that the free energy distribution corresponding to a fragment extending from position -148 to 51 in the E. coli sequences is appreciably different from the energy distribution calculated in randomly selected windows, at a significance level as high as 0.0001. A comparison of free energy distribution at position -20 (corresponding to the promoter region) with distributions at positions -200 (corresponding to the region upstream of promoter region) and +200 (corresponding to the coding region) is shown in Figure -10).In order to get a better insight into the stability feature, we carried out a detailed analysis of This difference in free energy and the stability of promoter regions as compared to that of coding and other non-coding regions can be used to search for the promoters. Based on this consideration, a new scoring function D(n) is defined, which will look for differences in free energy of the neighbouring regions of position n:D(n) = E1(n) - E2(n)where,E. coli) and the D value represents the free energy difference in the two neighbouring regions. A stretch of DNA is assigned as promoter only if the average free energy of that 50 nt region (E1) and difference in free energy as compared to its neighbouring region (D) is greater than the chosen cut-offs. The protocol followed to calculate the true and false positives and hence sensitivity and precision is presented in the form of a flowchart in Figure Thus, E1(n) and E2(n) represent the free energy (see METHODS) average in the 50 nt region starting from nucleotide n and neighbouring 100 nt region starting from nucleotide n+99, respectively. The E1 value represents the basal energy level, which is characteristic of the given bacterial genome . These cut-off values were then applied to a second set consisting of remaining 127 sequences. The sensitivity and precision values calculated for the first and second set match very well. We also found that very similar results can be obtained when we use the whole dataset  as compared to 0.109 precision (sensitivity = 0.443) achieved by NNPP. It can be seen that, for similar sensitivity levels, the precision achieved by our method (~0.7) is higher as compared to both TLS-NNPP and NNPP , by combining this method with additional information such as distance between TSS and translation start site (TLS), has been published . With thE. coli [Presence of high densities of promoter like signals in the upstream region of TSS may be one of the reasons why pattern matching programs result in low level of precision. This has been shown recently by a systematic analysis of sigma70 promoters from E. coli . In thiset al. [E. coli. This method is based on sequence as well as expression data, but requires prior knowledge of coordinates of every ORF in the genome. We would like to emphasize here that our method is different from other methods in that it is independent of any such prior knowledge about the test gene or the organism and hence holds promise as being useful for promoter prediction in a newly sequenced genome.Recently Bockhorst et al.  proposedet al. [et al. showed that a 30 % reduction of false positives can be achieved by use of physical properties, such as DNA bendability, in addition to other sequence properties of promoters. Interestingly, our method which also uses a physical property gives much smaller number of false positives as compared to Ohler et al.'s method. .The eukaryotic promoter prediction method proposed by Ohler et al.  is also Another vertebrate promoter prediction program, 'Promfind'  identifiE. coli RNA genes reported on the NCBI site [et al. [In addition to protein coding genes there are genes present for the non-coding RNAs (ncRNAs), which play structural, regulatory and catalytic roles. It is a difficult task to find out ncRNA genes in a genome because unlike protein coding regions they lack open reading frames and also they are generally smaller in size. In addition, it is also difficult to do a homology sequence search as only the structure of ncRNA is conserved and not the sequence. There are around 156 CBI site  and in a [et al.  recentlyEscherichia coli. Hence, we also tested our method using the promoter sequences from 1) the A+T-rich bacteria, Bacillus subtilis and 2) a G+C rich bacteria such as Corynebacterium glutamicum. Figure Escherichia coli. It can be clearly seen that, at present our method performs optimally for the Escherichia coli promoters and also performs quite well in case of Bacillus subtilis. The prediction accuracy in case of Corynebacterium glutamicum promoters is not as good as that for the other two classes of promoters. However, it should be noted that the number of experimentally determined Corynebacterium promoters is much smaller as compared to other two bacteria and a larger dataset is required to arrive at any firm conclusion.Finally, it is very important to see whether the method works equally well for other organisms which have genome compositions substantially different from that of It has often been suggested that use of certain properties of promoters, other than just the sequence motifs, which can distinguish promoters from other genomic regions, could significantly improve the gene prediction methods. Although the lower stability of promoter regions as compared to non-promoter regions has been reported previously, this observation was not incorporated into a promoter prediction program. We have been able to successfully use the differential stability of promoter sequences to predict promoter regions. Our method performs better as compared to currently available prokaryotic prediction methods and is also moderately successful in predicting RNA and bacillus promoter regions. The method certainly needs to be further improved to reduce the number of predicted false positives. This can be achieved by combining the approach presented here, with the earlier reported sequence analysis methods. Such a composite method will also help in pinpointing the TSS within the promoter region identified by our method.E. coli promoters, 89 B. subtilis promoters and 28 C. glutamicum promoters.All the promoter sequences used in this study are 1000 nt long, starting 500 nt upstream (position -500) and extending up to 500 nt downstream (position +500) of the TSS. In order to avoid having multiple TSS in a given 1000 nt sequence, we have excluded all the transcription start sites which are less than 500 nt apart. Our promoter set has 227 Escherichia coli promoter sequences, which were taken from the PromEC dataset [Escherichia coli genome sequence (NCBI accession no: NC_000913).We tested our algorithm using the  dataset . The ProBacillus subtilis promoters were obtained from the DBTBS database [The transcription start sites for database . The reqCorynebacterium glutamicum promoters is carried out on a set of promoters compiled by Pàtek et al. [Analysis of k et al.  based onThe transcription start positions of RNA transcription units are obtained from the ecocyc dataset. In this set, both computer predicted as well as experimentally determined transcription start sites, are included. In total, we have 7 rRNA TUs, 33 tRNA TUs and 2 TUs of other RNAs.o37) corresponding to the melting transition of an 'n' nucleotides (or 'n-1' dinucleotides) long DNA molecule, from double strand to single strand is calculated as follows:The stability of DNA molecule can be expressed in terms of free energy. The standard free energy change . For each window, the free energy is calculated as given in the above equation and the energy value is assigned to the first base pair in the window. The energy values corresponding to the 10 unique dinucleotide sequences are taken from the unified parameters proposed recently [Since our analysis involves long continuous stretches of DNA molecules, in our calculation we did not consider the two terms, ΔGrecently ,35.E. coli sequences ranging from -500 to +500, was compared to the distribution in a randomly selected set. For this comparison, we followed a similar procedure as adopted by Margalit et al. [The free energy distribution at a given position, in the 1000 nt t et al. . The ranWe compared the free energy distribution at position -20 (with respect to TSS) with the distributions at the positions -200 and +200 using Kolmogorov-Smirnov two sample test .®.All the calculations related to the statistical tests were carried out using MATLAB 6.0viz. NNPP and Staden's method. NNPP program is available at [The promoter predictions were also carried out using two other methods lable at . All theThe implementation of Staden's method was carried out as described in ,37. The In case of NNPP as well as Staden's method, the true and false positives were scored as in case of our method Figure , with a The sensitivity and precision for the predictions are calculated using the following formulae:AK performed the analysis, evaluated the results, and drafted the manuscript. MB suggested the problem, helped with evaluation of the results and the manuscript, also provided mentorship. All authors read and approved the final manuscript.


Armored scale insects are unusual in that a part of their bodies is genetically distinct from the rest. This extraordinary phenomenon challenges the notion of identity I am a clone. That is, I am a colony of cells that developed from a single fertilized egg cell. Most animals are clones like me. It is a slight oversimplification to say that all of an animal's cells are genetically identical to each other. Some cells have mutations. In mammals, some cells (red blood cells) lack a nuclear genome entirely. Some cells have viruses—and when it's in a cell, a virus is basically a gene—that other cells lack. But a typical animal is a clone in the sense that all its cells arise from that single fertilized egg cell.Not all animals, however, are clones. Sometimes two tiny embryos developing inside their mother will fuse together into a single embryo and continue developing. The resulting animal is not a clone, but a chimera: a conglomeration of two different cell lineages into a single organism. Some species of monkeys (marmosets) typically have chimeric blood, from having shared a blood supply with a twin in utero , and rarObligate chimerism—the presence of two genetically distinct cell lineages in every individual at each life stage—is found in a few families of scale insects, but nowhere else in nature. The avid naturalist wants to understand this sort of deep oddity for its own sake, but such understanding might have broader implications as well. For instance, although humans are not usually chimeras, we do have a quasi-chimeric phase in our life cycle: pregnancy. Some diseases of pregnancy are apparently due to conflicts between the genetically nonidentical tissues of mother and fetus . And theIn all sexual animals and plants, production of an egg cell involves meiosis, the complex cellular process  whereby one diploid nucleus (with two copies of each chromosome) becomes four genetically different haploid nuclei (each with one copy of each chromosome). Only one of these four haploid nuclei becomes the egg cell (oocyte). In ordinary animals, the other three nuclei (the polar bodies) degenerate—they never divide again and are lost or destroyed—and the oocyte is the single maternal cell that  develops into the embryo. But in armored scales, the polar bodies fuse together into a triploid cell (with three copies of each chromosome), and this triploid cell also winds up in the embryo . The triNow the story gets even stranger. If the individual is a male, then the genetic difference between his bacteriome and the rest of his tissues becomes even greater as he develops. This is because most armored scale insects have an unusual genetic system called embryonic paternal genome elimination . In maleWhat could possibly be going on here? Why should scale insects, of all creatures, have obligate chimerism involving activated polar bodies? Essentially, we have no idea, largely because no one has even ventured a serious guess. When the phenomena were discovered, early in the 20th century, the theoretical tools for making sense of them were unavailable. One such tool is W. D. less closely related than two siblings would be, because the polar bodies are enriched for chromosome regions not present in the oocyte.Consider the special theoretical difficulty posed by chimerism between tissues derived from the oocyte and those derived from the polar bodies ejected by it during meiosis. Two siblings will typically exhibit some degree of sibling rivalry—their interests are not identical. If an individual were a chimera comprising full-sibling tissues , there might be conflict between the two nonidentical cell lineages, as there is between the tissues of a mother mammal and her fetus  during pregnancy . This maat all . Crossing over prevents this, creating a mosaic of related and unrelated chromosome regions between the products of the first meiotic division and uncertain relationships between the final four meiotic products. Nonetheless, the consistently depressed relatedness between the oocyte and the polar bodies may help to explain why polar bodies are almost always eliminated—sibling rivalry might be even greater if some siblings were derived from each other's polar bodies.If there were no crossing over between homologous chromosomes during meiosis, then the first meiotic division would consistently separate the chromosomes derived from the mother's mother from the chromosomes derived from the mother's father, producing two cells that are not related to each other So how and why did two families of scale insects tame and domesticate their potentially fractious polar bodies, rather than killing them like normal animals do? There are at least three lines of thinking that seem promising for unraveling this mystery.There are interesting parallels between, on the one hand, the chimerism seen in armored scale insects and, on the other hand, the eusociality  seen in ants and honeybees. In ants and honeybees, sterile individuals (workers) provide nutrition to their potentially fertile siblings. In armored scale insects, a genetically distinct but ultimately sterile cell lineage (the bacteriome) provides nutrition to its potentially fertile “sibling” cell lineage —though, of course, polar body-derived cells are “sibling” in a strange special sense. Like ants and bees, armored scale insects are effectively haplodiploid: males transmit only the chromosomes they inherited from their mother, and all of a male's sperm are identical to each other. This “clonality” of sperm boosts the relatedness between sisters, and This temptation increases when we consider another case from the old cytogenetics literature of apparent histological eusociality, though not of true permanent chimerism. This occurs in a few families of parasitoid wasps , in whicBut although it is tempting to conclude that haplodiploidy plays the same role in promoting histological eusociality as it does in promoting organismal eusociality, the temptation should probably be resisted. In the case of the parasitoid wasps, the polar body-derived tissue has no paternal genome, so the clonality of sperm cannot boost its relatedness to anything. In the case of armored scale insect chimerism, the bacteriome does have a copy of the paternal genome, and that copy is identical to the paternal genome in the rest of the embryo, so in that sense the two tissues do have a high relatedness similar to the high relatedness of full siblings under haplodiploidy. But the scale insect bacteriome gets its copy of the paternal genome directly from the embryo, so the clonality of sperm (the source of elevated relatedness between haplodiploid sisters) apparently has nothing to do with it. Some other explanation is probably needed.less related to you, on average, than your siblings are, they are more related to your siblings  than they are to you! Perhaps the polar bodies function to somehow enforce some maternal or family interest, nipping in the bud some sibling rivalry that would otherwise suppress the family's fitness. In haplodiploid groups, females are more closely related to their full sisters (with whom they share three-quarters of their gene copies) than to their brothers (with whom they share only one-quarter of their gene copies). So if there is competition between siblings for resources, females are expected to behave more antagonistically towards brothers and more cooperatively towards sisters. In contrast, a female is equally related to a son as she is to a daughter . These asymmetries in relatedness lead to struggles in haplodiploid social groups, with a mother seeking to direct more resources towards sons and with sisters seeking to direct more resources away from their brothers and towards each other , after which she settles in one spot permanently. The male is slightly more mobile, having a motile  adult form. Nonetheless, (1) the low motility of females, and the fact that they live mostly on long-lived woody plants, means that maternal kin may interact over long timescales, as in social insects; (2) some scale insects appear to make relatively sophisticated social decisions about where to settle, settling nearer to (and thus possibly competing more closely with) non-kin than kin ; (3) altSimilar manipulation of intersibling interaction might be going on in the case of the parasitoid wasps that have polar body-derived membranes around their eggs. Sometimes these wasps lay a single unfertilized  egg and a single fertilized  egg into the same host. Both eggs divide to form embryos, which divide into a clone of many embryos. Some of the embryos become sterile “precocious” larvae that can apparently attack other larvae trying to use the same host—including, potentially, their own siblings . Here isThe endosymbiotic bacteria that dwell in the scale insects' bacteriomes are maternally inherited. Thus, from the perspective of the bacteria, male insects are deadends. Many maternally inherited bacteria have evolved to manipulate the hosts' genetic system for their own advantage. Some bacterial lineages induce parthenogenesis or feminize males. Bacteria may even evolve to suicidally kill male embryos that they find themselves in, if the death of the male frees up resources that his sisters can use . In ordeScale insects and their bacteriomes challenge our notion of what an individual is. Is a scale insect's bacteriome a kind of sibling? Is it half sibling, half self? Is it a sterile slave, under control? Is it an extension of the mother, exerting control? In all other organisms, chimeras are temporary and unstable. How have scale insects suppressed the conflicts that normally tear chimeras apart? To approach such questions, we'll have to revive the empirical study of scale insect bacteriomes, combining approaches from recent studies of aphid bacteriomes  and of h