Increasing antimicrobial resistance among the key pathogens responsible for community-acquired respiratory tract infections has the potential to limit the effectiveness of antibiotics available to treat these infections. Since there are regional differences in the susceptibility patterns observed and treatment is frequently empirical, the selection of antibiotic therapy may be challenging. PROTEKT, a global, longitudinal multicentre surveillance study, tracks the activity of telithromycin and comparator antibacterial agents against key respiratory tract pathogens.In this analysis, we examine the prevalence of antibacterial resistance in 1,336 bacterial pathogens, isolated from adult and paediatric patients clinically diagnosed with acute bacterial sinusitis (ABS).S. pneumoniae isolates were susceptible to penicillin, cefuroxime, and clarithromycin respectively. Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of S. pneumoniae isolates  while 99.5% and 95.5% of isolates were susceptible to telithromycin and amoxicillin-clavulanate, respectively. In total, 88.2%, 87.5%, 99.4%, 100%, and 100% of H. influenzae isolates were susceptible to ampicillin, clarithromycin, cefuroxime, telithromycin, and amoxicillin-clavulanate, respectively. In vitro, telithromycin demonstrated the highest activity against M. catarrhalis .In total, 58.0%, 66.1%, and 55.8% of in vitro activity of against pathogens commonly isolated in ABS, together with a once daily dosing regimen and clinical efficacy with 5-day course of therapy, suggest that telithromycin may play a role in the empiric treatment of ABS.The high  The incidences of both the acute and chronic forms of sinusitis have been increasing, and between 10 and 15% of the population of central Europe are affected annually -4. AcuteS. pneumoniae is the most common pathogenic bacterium responsible for ABS, isolated in 30–50% of cases, followed by H. influenzae, isolated in 20–40% of cases. Moraxella catarrhalis is isolated in 5–10% of cases, beta haemolytic streptococci in less than 5%, and Staphylococcus aureus in less than 10% although it is often found co-infecting with other bacteria [Treatment options for ABS are controversial as up to 40% of patients recover spontaneously, however, studies have shown that treatment with an antibacterial reduces the time to recovery from sinusitis, improves symptoms, and helps to prevent complications ,12. GuidSecond line, or alternative antibacterial agents of choice, are clarithromycin or second-generation cephalosporins such as cefuroxime and cefpodoxime, third-generation cephalosporins such as cefdinir, and trimethoprim-sulphamethoxazole ,5,16,17.in vitro activity and demonstrated clinical efficacy against bacterial pathogens causing community-acquired respiratory tract infections (RTI's) [in vitro activity against the common community-acquired RTI pathogens [However, due to increasing levels of resistance in bacterial respiratory tract pathogens to these commonly used antibacterials (particularly the rapid emergence of penicillin-and macrolide-resistant strains of pneumococci), new agents are required that have high  (RTI's) -22. Teliathogens . Clinicaathogens -26.The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is a longitudinal, global multicentre surveillance study designed in part to determine the activity of telithromycin against community-acquired RTI isolates, in relation to the frequency of prescribing, in the regions where the study is conducted . The aimDetailed study design, including patient selection and methodology for isolate identification and storage in the PROTEKT study has been described previously . The isoS. pneumoniae and S. aureus, ≤ 1 μg/ml is susceptible, 2 μg/ml is intermediate, and ≥ 4 μg/ml is resistant; for Haemophilus influenzae, ≤ 4 μg/ml is susceptible, 8 μg/ml is intermediate, and ≥ 16 μg/ml is resistant [MIC susceptibility status was determined, using the National Committee of Clinical and Laboratory Standards (NCCLS) breakpoints, at a central laboratory  from a panel of existing and new antibacterials, using the NCCLS broth microdilution method and lyophilised microtitre plates  . NCCLS besistant .2 test.Statistical analysis was performed using a χn = 652), North America (n = 14), Latin America (n = 207), Asia (n = 464), Eastern Europe (n = 68), Australia (n = 2), and South Africa (n = 126) in the PROTEKT study from years 2000–2001 and 2001–2002 for analysis of the susceptibility of bacterial pathogens isolated from patients with acute sinusitis. Gender distribution was 52.2% male (695 patients), 46.7% female (624 patients); gender was not provided for 1.3% of patients. Almost two thirds (66.3%) of patients were in the 0–12 year age group, one third (29.7%) in the 13–65 year age group, 2.7% in the over 65 year age group and age was not specified in 1.3% of patients. S. pneumoniae was the pathogen most frequently isolated (47.9% of isolates) followed by H. influenzae (24.6% of isolates) . Of note, 3 isolates of S. pneumoniae were non-susceptible to telithromycin . This represented 0.5% of isolates, a value that is significantly (p < 0.001) lower than those obtained by erythromycin (44.1%), clarithromycin (44.2%) and cefuroxime (33.9%).Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of H. influenzae isolates, 34 (10.3 %) were positive for β-lactamase production. All isolates of H. influenzae were susceptible to amoxycillin-clavulanate and telithromycin with an MIC90 of 1 and 2 mg/L, respectively. Amoxycillin-clavulanate and telithromycin were more potent and had greater activity than clarithromycin . This activity was comparable to azithromycin .Of the 329 S. aureus isolated from the total number of specimens was small (116/1366 isolates), telithromycin was as efficacious as comparators. Of the 116 isolates, 11 were resistant to methicillin (MRSA) and 105 were methicillin susceptible (MSSA). Ninety-nine (94.3%) of the MSSA isolates and 5 of the 11 MRSA isolates were susceptible to telithromycin. Of note, all of the S. pyogenes isolates were inhibited by ≤ 1 mg/L telithromycin, despite 17.7% resistance to erythromycin and clarithromycin.Although the number of M. catarrhalis with an MIC50 of 0.06 mg/L and MIC90 of 0.12 mg/L. β-lactamase production was detected in 97.6% of these isolates.Telithromycin was the most potent antimicrobial against in vitro activity against bacterial pathogens isolated from a large, globally distributed population of patients diagnosed with ABS. Telithromycin was the most active and potent agent against all isolates of the pathogens isolated from patients with ABS with 99.4% of isolates susceptible. Not surprisingly, high levels of penicillin resistance, macrolide resistance, and combined penicillin and macrolides resistance were prevalent in S. pneumoniae although prevalence varied widely between geographical regions.The data in this analysis demonstrates that telithromycin has high H. influenzae and M. catarrhalis found in the present study demonstrate compromised in vitro efficacy of amoxicillin against these isolates. Although the cephalosporins  showed high activity against H. influenzae (including beta-lactamase positive strains), resistance to these agents was high in S. pneumoniae: >30% for cefuroxime and cefpodoxime – cefdinir was not tested against pneumococci in PROTEKT, however susceptibility is usually similar to the other cephalosporins reported here [Amoxicillin has been the treatment of choice in ABS because of its general effectiveness, safety, tolerability, low cost and narrow spectrum . The higS. pneumoniae of 44.1% to erythromycin, azithromycin, and clarithromycin was found. Trimethoprim-sulfamethoxazole activity was low for S. pneumoniae (56.3% susceptible) and decreased for H. influenzae (84.5% susceptible).Similarly, macrolides are prescribed in various countries for ABS and an overall resistance rate for Respiratory fluoroquinolones are recommended second-line treatment options in some countries (references needed to support this statement). However, recent evidence suggests that resistance to fluoroquinolones is rapidly developing in pneumococci and other pathogens  High rates of clinical cure and bacteriological eradication have been demonstrated using telithromycin against sinus isolates of S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus [The high . aureus -26. 2) T. aureus -39. 3) T. aureus ,41. This. aureus . 5) Teli. aureus ,45.S. pneumoniae isolates from patients with community-acquired pneumonia, they may then only send H. influenzae from patients with ABS to fulfill their quota for this organism. Thus, the potential exists to over or under estimate the prevalence of a species in a particular disease. A further limitation of this study is it is restricted to the major bacterial pathogens causing sinusitis and does not therefore assess anaerobic bacteria, which are also known to be involved in this disease. However, a recent study of sinus puncture specimens demonstrated that telithromycin had good in vitro activity against anaerobes involved in sinusitis [Although this study provides valuable information on the overall antimicrobial profile of bacteria causing ABS care should be taken when interpreting data related to specific demographics. A major limitation of this study, inherent to most surveillance studies, is the requirement for collecting centers to fulfill a specified quota of isolates over a defined time period (1 year). If, for instance, 1 center manages to fulfill their quota for inusitis .Streptococcus pneumoniae and Haemophilus influenzae. Additionally, there are regional differences in the susceptibility patterns observed and, as therapy is usually empirical, choosing an effective therapy can be challenging [The inclusion of nasopharyngeal specimens is a potential limitation of this study and the higher rate of resistance compared to sinus aspirates may indicate some isolates were nasopharyngeal flora rather than pathogens. However, the difference in resistance prevalence between nasopharyngeal specimens and sinus aspirates was not great for any species/antibiotic combination, and assuming the majority of isolates were the responsible pathogen, significant bias of resistance patterns is unlikely. The treatment of ABS is complicated by a difficulty in establishing the causative pathogen(s). Sampling of infected fluid using sinus puncture is a painful and rare procedure . Nasophallenging ,20,49.in vitro activity against S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus respiratory pathogens commonly isolated in ABS. It is as active as or more active than antibacterial agents that are currently used in this clinical setting. The development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the in vitro efficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained telithromycin currently offers a useful therapeutic option in the treatment of AS.In summary, the data presented here demonstrates that telithromycin has good JD, RCa and RCo reviewed the data and provided clinical and microbiological interpretation and discussion. DF and DJF participated in the design of the study, supervised the scientific testing, and provided data analysis, microbiological interpretation and discussion. All authors drafted the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.The M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows.MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.Collectively, these results demonstrate that while the 19-kDa protein from  Mycobacterium avium subsp. paratuberculosis (referred to hereafter as M. avium subsp. paratuberculosis) and induces a chronic enteritis in ruminants. The disease signs include weight loss, diarrhea, and decreased milk production. In the United States alone the economic burden of Johne's disease is estimated at over $200 million in lost annual revenue to the dairy industry [M. avium subsp. paratuberculosis [Paratuberculosis (Johne's disease) is caused by industry . Prevalerculosis . Neonatarculosis ,5. Durinrculosis . Vaccinerculosis -9.M. tuberculosis genome that possess a characteristic amino-terminal acylation motif [Mycobacterium tuberculosis is immunodominant in both mice [+ T cell proliferation as well as the release of IL-2, IFN-γ, and IL-12 [M. tuberculosis 19-kDa protein inhibits innate immune responses, such as the release of TNF-α, IL-6, and IL-10 from macrophages, but does not affect antibody binding [M. tuberculosis-infected monocytes [M. tuberculosis residing within the phagolysosomal compartment [M. bovis, M. avium, and M. intracellulare but are absent from M. phlei, M. smegmatis, M. fortuitum, M. gordonae, and M. leprae [Lipoproteins have long been considered immunomodulators and mycobacteria are especially rich in these post-translationally modified proteins. There are approximately 100 open reading frames identified in the oth mice -13 and hoth mice ,15 and hnd IL-12 ,17. Acyl binding -20. The onocytes  as well onocytes . Finallyonocytes ,23. Expepartment . Homolog. leprae .M. avium subsp. paratuberculosis proteins involved in metabolism, cell wall synthesis, macrophage entry and survival, disease pathogenesis, or host immune evasion. However, several antigens have recently been identified and their immunogenicity examined by serodiagnostic and/or lymphocyte stimulation assays [M. avium subsp. paratuberculosis recently defined [M. avium subsp. paratuberculosis enable us to select and characterize genes of interest. A major goal of this laboratory is to define a complete catalog of immunodominant antigens in M. avium subsp. paratuberculosis. With the immunostimulatory capabilities of the M. tuberculosis 19-kDa antigen in mind, the objective of this study was to determine if the 19-kDa protein of M. avium subsp. paratuberculosis possessed a similar capacity. In this study, the 19-kDa lipoprotein from M. avium subspecies paratuberculosis was cloned, expressed, and characterized. In addition, the purified recombinant protein was used to assess cellular immune responses in subclinically infected cattle as well as humoral immune responses in cattle with clinical Johne's disease.Despite decades of research, little is known about the n assays -30. With defined , all proM. avium subsp. paratuberculosis genome project as MAP0261c. Comparison of amino acid sequences from other species of mycobacteria show that this gene product is conserved. Sequence alignment shows the N-terminal half is more variable and the region between amino acids 99 and 123 is highly conserved  detected a signal peptide generated from a putative cleavage site between amino acids 34 and 35, to expose an N-terminal serine -19 kDa fusion protein that was affinity-purified from E. coli lysates. MBP-19 kDa was analyzed by SDS-PAGE  that detects the MBP affinity tag . By comparison, sera from 9 of 14 infected cattle reacted specifically with the 19-kDa protein, but not MBP alone. Antibody reactivity to the 19-kDa protein from 3 clinical cows is shown in Figure The M. avium subsp. paratuberculosis sonicated whole cell lysate (WCL), MBP, or MBP-19 kDa and IFN-γ production was assayed by ELISA. These cattle were selected from a larger group because they showed no IFN-γ stimulation in response to MBP alone. IFN-γ production in response to WCL stimulation allowed for the segregation of infected cattle into three groups: suspect, positive, and high positive. After subtracting the IFN-γ responses of non-stimulated cells, suspect animals had less than 0.1 absorbance units of IFN-γ production, positive animals had 0.1 – 0.3 absorbance units of IFN-γ production, and high positive animals had more than 0.3 absorbance units of IFN-γ production. IFN-γ responses by blood mononuclear cells from infected cattle exceeded responses from control cattle for both the WCL and MBP-19 kDa  or WCL, which may not be specific for M. avium subsp. paratuberculosis. Recent completion of the M. avium subsp. paratuberculosis genome has already advanced efforts to identify novel antigens [M. avium subsp. paratuberculosis 19-kDa protein, as well as assess its immunostimulatory capabilities in cattle.A majority of the research on individual mycobacterial proteins has been performed in antigens ,34. FurtM. avium subsp. paratuberculosis can be readily overexpressed as a fusion protein in E. coli. This is not true for many other proteins encoded by M. avium subsp. paratuberculosis [E. coli. Previous studies have suggested the M. tuberculosis 19-kDa protein undergoes posttranslational modification by the addition of fatty acids to form a lipoprotein [E. coli host. It is unclear whether posttranslational modification of this protein would affect its immunological activity. The recombinant antigen was detected by sera from cattle with Johne's disease; however, it was not as strong a stimulator of proliferative T-cell responses as has been reported for its counterpart in M. tuberculosis [M. tuberculosis was shown to induce both cellular and humoral immune responses from mice and humans [In this study, we show that the 19-kDa protein of rculosis  and suggoprotein . Althougrculosis . Furtherd humans ,14. ThesM. avium subsp. paratuberculosis-infected cattle are biphasic, with IFN-γ responses detected early and antibody responses detected late in infection. However, evidence suggests that an unknown M. avium subsp. paratuberculosis protein can be detected by antibodies from cattle just 3 weeks after infection [M. avium subsp. paratuberculosis (WCL), and IFN-γ production was measured. Results from this study suggest that while the 19-kDa protein is a stimulator of IFN-γ production, it is not as potent when compared to WCL. Additionally, we found that a majority (9 of 14) of infected cattle produced antibodies to the 19-kDa protein, as determined by immunoblot analysis. By comparison, sera from the majority of control, non-infected cattle (8 of 9) did not react to the 19-kDa protein. The single non-infected cow that did show reactivity to the MAP0261c gene product may be attributed to exposure of environmental mycobacteria such as M. avium subsp. avium, which has a similar protein .It is generally accepted that cellular and humoral immune responses of nfection . As a meM. tuberculosis 19-kDa protein was reported to contain a 21 amino acid N-terminal signal peptide [M. avium subsp. paratuberculosis identified a putative 34 amino acid N-terminal signal sequence. Furthermore, the computer algorithm PSORTb predicts a putative signal sequence, but it cannot determine if the protein is actually secreted. Antibodies will be produced against the recombinant MBP-19 kDa protein to determine if the protein is secreted by M. avium subsp. paratuberculosis.The  peptide , howeverM. avium subsp. paratuberculosis to identify infected cattle but should not be used as a "stand alone antigen" in new diagnostic assays.The results from this study show that the recombinant 19-kDa protein stimulates a weak host immune response in infected cattle. The 19-kDa protein may be used in conjunction with other antigens from M. avium subsp. paratuberculosis strain 19698-1974  was grown in Middlebrook 7H9 liquid media (pH 6.0) supplemented with 10% oleic acid albumin dextrose complex , 0.05% Tween 80 (Becton Dickinson Microbiology), and 2 mg/ml mycobactin J . M. avium subsp. paratuberculosis cultures were grown to log phase at an optical density 540 nm (OD540) of 0.4, at 37°C without shaking. Escherichia coli DH5α cells were routinely grown in Luria-Bertani (LB) broth or LB agar plates at 37°C supplemented with ampicillin (100 μg/ml) for selection.M. avium subsp. paratuberculosis. The animals used in IFN-γ experiments were placed in four groups consisting of 9 non-infected healthy cows, 9 suspect subclinical cows (IFN-γ responses < 0.1), 6 positive subclinical cows (IFN-γ responses 0.1 – 0.3), and 6 high-positive cows (IFN-γ responses > 0.3). The non-infected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3- to 5-year period. In addition, these animals were negative on all immunological assays (i.e. ELISA and IFN-γ production) performed during that period. Subclinical cattle  were characterized by shedding less than 10 CFU/g of feces and were intermittently positive by IFN-γ assays (response > 0.1) performed quarterly over a 3- to 5-year period. The institutional Animal Care and Use Committee approved all animal procedures described in this study.The Johne's Disease Research Project at the National Animal Disease Center has a repository of sera from cattle that were euthanized with clinical signs of Johne's disease, which included shedding, weight loss and diarrhea. Fecal samples from each of these clinical cattle were found to contain more than 100 CFU per gram of feces as determined by colony counts on Herrold's egg yolk media (HEYM) agar slants by standard culture methods . Sera frM. tuberculosis, M. bovis, M. avium and M. intracellulare were obtained from the NCBI nucleotide sequence database. The sequences were assembled and compared using MegAlign software .The nucleotide sequences for the 19 kDa coding regions from M. avium subsp. paratuberculosis 19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector . To amplify the 19-kDa coding region from M. avium subsp. paratuberculosis, primers were designed directly from the MAP0261c sequence. MAP0261c was amplified with Expand High Fidelity PCR system using the primers 19-kDa-pMal5' (5'-GCGCCAGCTGACGATCGCGGTCGCGGGCGCGGC-3') and 19-kDa-pMal3' (5'-GCGCAAGCTTCAGGTCACATCGATCTCGAAC-3'). The 19-kDa-pMal5' and 19-kDa-pMal3' primers contained Pvu II and Hind III restriction sites (underlined) for cloning, respectively. This primer set amplified the 19-kDa coding sequence minus the N-terminal 4 amino acids and the C-terminal 3 amino acids. The pMal-c2 vector was digested with Xmn I and Hind III and the PCR amplicon was digested with Pvu II and Hind III. The two products were ligated overnight at 12°C with T4 DNA ligase , which resulted in an in-frame fusion between the vector-encoded malE gene and a majority of the 19-kDa gene. The resulting recombinant plasmid, designated pMal-19-kDa, was transformed into E. coli DH5α competent cells. Recombinant clones were selected by plating on LB-ampicillin plates overnight at 37°C. Individual clones were picked and inserts were confirmed by DNA sequencing. The resulting fusion protein was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs). A detailed expression and purification protocol has been published previously [A maltose binding protein (MBP) fusion of the eviously . ExpressE. coli lysates expressing MBP-19 kDa were prepared as previously described [escribed . SDS-PAGM. avium subsp. paratuberculosis sonicate (WCL), 10 μg/ml of MBP, or 10 μg/ml of MBP-19 kDa. The WCL was prepared by sonication of bacilli and centrifugation exactly as described previously [2 humidified atmosphere. Plates containing blood-antigen samples were centrifuged at 500 × g for 15 minutes and the plasma was harvested from each well. Plasma samples were frozen at -20°C until being analyzed for IFN-γ concentration by enzyme-linked immunosorbent assay (ELISA) using a commercial kit  as recommended by the manufacturer. Samples were analyzed in duplicate and were determined to be positive for IFN-γ production if the absorbance of the stimulated sample  was 0.1 units greater than the absorbance of the nonstimulated well for that animal. This classification of IFN-γ positive samples has been previously reported by our laboratory as well as others [Blood was collected from the jugular vein of subclinically-infected cattle into sodium heparin vacutainer blood collection tubes. One ml aliquots of whole blood from each animal were plated into 4 wells of 24-well culture plates and cultured alone (non-stimulated) or with 10 μg/ml of eviously . Blood-as others ,42.ANOVA and unpaired t tests were performed to analyze the IFN-γ stimulation data. Analyses were performed to compare average stimulation of control, non-infected cattle to the infected cattle groups . Differences were considered significant when P < 0.05.JFH carried out all the experiments and drafted the manuscript as part of his PhD dissertation. JRS provided advice, participated in its design and coordination and helped edit the manuscript. JPB conceived of the study, participated in its design and helped to draft the manuscript.


Multiple instruments exist to measure dementia behaviors, but the nursing staff perspective on those behaviors and their level of burden has not been well measured. The goal of this study was to examine the psychometric performance of the Modified Nursing Care Assessment Scale (M-NCAS), a 28-item nurse rating of burden associated with care for institutionalized individuals with dementia. Nurses rate items in terms of extent to which the behavior or characteristic is present ("attitude" domain), and extent to which it is a burden ("strain" domain).Data from 282 patients enrolled in a 12-week, double-blind, randomized clinical trial comparing risperidone treatment to placebo was used to evaluate M-NCAS item performance, internal consistency reliability, and construct validity. Empirical subscales were identified via exploratory factor analysis (EFA).Four poorly-performing items were deleted from further analyses. EFA identified 3 "attitude" subscales and 5 "strain" subscales. Cronbach's alphas were 0.65 and above. Correlation with the Cohen-Mansfield Agitation Inventory and the BEHAVE-AD, clinical ratings of dementia behaviors, were low to moderate.The M-NCAS provides a valid and reliable means of obtaining care burden ratings from formal caregivers in long-term care, and provides a method for evaluating dementia interventions from the perspective of nursing staff. Caring for the institutionalized dementia patient can be challenging for nursing staff ,2. PersoThere are measures relevant to the burden of paid/formal caregivers , but most focus on such overt behaviors as "forgetting what day it is" or "pull away" only  ,10 was dThe M-NCAS was used in a clinical trial of an atypical antipsychotic, risperidone, for treatment of nursing home patients with behavioral and psychological signs and symptoms in dementia. This preliminary investigation describes the psychometric evaluation of the M-NCAS using data from the clinical trial; thus, it is a post-hoc assessment of reliability and validity. Assessment of psychometric properties included exploration of item performance, subscale assignment, and assessment of reliability and validity based on the available data from the clinical trial.The psychometric evaluation used data from the baseline assessment of a multi-site, double-blind, placebo-controlled trial of risperidone in the treatment of behavioral and psychological symptoms among institutionalized patients with dementia. Fourteen sites throughout Australia and New Zealand recruited patients from the various nursing homes associated with each site. Patients were included in the clinical trial if they were 55 years of age or older, residing in a nursing home environment for one month, with a DSM-IV diagnosis of either dementia of the Alzheimer's type with behavioral disturbance, vascular dementia with behavioral disturbance, or mixed dementia, had scores less than or equal to 23 on the Mini-Mental Status Exam (MMSE) , and metThe Behavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD), the CMAI, and the M-NCAS were administered to the nurse carers of the individual patients by the investigator or site coordinator at baseline, week 4, week 8, and week 12 . Nurse carers could have more than one patient participating in the trial, and they completed questionnaires for each individual patient.The M-NCAS is was adapted from the original NCAS instrument developed in Sweden, and contains 32 items based in part on the 21 original items of the NCAS. Additional items were selected based on comments made by long-term care nurses regarding their experience caring for dementia patients with dementia in long-term care facilities, and added to make the instrument more comprehensive.The M-NCAS contains 32 items, with two domains per item: one addresses the occurrence and intensity of the behavior, in which the staff members indicate the extent to which they agree that the patient exhibits the behavior (the "attitude" domain); and one which addresses staff rating of the difficulty of coping with the behavior (the "strain" domain). Responses are measured on a four-point Likert-type scale . Lower scores are better for both domains. Domain total and subscale scores are calculated separately; total and subscale scores are calculated as an average of the individual item scores. The M-NCAS was translated from the original Swedish to English using standard forward and backward translation techniques.For subscale analyses, if < 50% of the scale items were missing, the scale was scored with the mean score of the non-missing items for that individual used to impute a score for the missing items. If > 50% of the items were missing, no scale score was calculated; the subscale score was considered missing. Total scores were calculated only if < 50% of the items were missing. Missing item criteria was based on that recommended by Ware et al. .The CMAI is a 29-item scale specifically developed to assess agitated and disruptive behaviors, as well as care-related problems, occurring in demented subjects residing in nursing homes. The scale measures the frequency of behaviors, and systematically assesses agitation. The scale's 29 activities are rated on a 7-point scale indicating the frequency of a particular activity . The activities are organized into 4 subscales: physical/aggressive, physical/non-aggressive, verbal/aggressive, and verbal/non-aggressive. The total aggression score is the sum of the physical and verbal aggression subscales. The total non-aggression score is the sum of the verbal and physical non-aggression subscales. A recall period of one week was used. The CMAI was used to assess the construct validity of the M-NCAS.The BEHAVE-AD is a 25-item instrument designed to assess the severity of behavioral disturbances in subjects with dementia, based upon the caregiver's observation and report of the subject's behavioral problems. It consists of 7 subscales: paranoid and delusion ideation, hallucinations, activity disturbances, aggressiveness, diurnal rhythm disturbances, affective disturbances, and anxieties and phobias. Symptoms are rated on a scale from 0 (absence of the symptom) to 3 (greatest severity). A global item is included, in which a single judgment is made as to how troubling or dangerous the subject's behavior has been to the caregiver. A recall period of one month was used. The BEHAVE-AD was used to assess the construct validity of the M-NCAS.We evaluated the item performance, scaling characteristics, reliability, and construct validity of the M-NCAS using baseline assessment data.Item performance was examined to identify the presence of items that may reduce the instrument's ability to detect changes over time, and to discriminate between groups. The characteristics of individual M-NCAS items examined were mean, minimum and maximum responses, percent missing, floor and ceiling effects, and item-total correlations. These data were used to determine if some items should be deleted for subsequent analyses . ExplorInternal consistency reliabilities of total and subscale scores were estimated using Cronbach's coefficient α. Data to examine test-retest reliability was not available.Correlations were determined for total and subscale scores for attitude and strain domains and the CMAI physical and verbal aggression and non-aggression subscales. Correlations were also calculated between M-NCAS scores and the total score, seven subscales, psychotic symptoms subtotal and global rating question of the BEHAVE-AD, and the CGI-S and CGI-C scores. Pearson correlations were used unless otherwise specified. Correlations were expected to be low to moderate (0.20 ≤ r < 0.40).All statistical analyses were conducted using SAS statistical software version 8.0. A significance level of 0.05 and 2-tailed tests were used unless otherwise noted. No adjustments were made for multiple comparisons.Two hundred and eighty-two patients had evaluable baseline data for the psychometric evaluation. The majority of patients were female (72.4%) and Caucasian (98.4%); 11.5% were between the ages of 65 and 74, while 87.1% were over age 74. Most subjects had a diagnosis of Alzheimer's Disease (59.1%), while 28.3% had vascular dementia and 12.5% had mixed dementia.Nurse carers were primarily Caucasian. Further demographic data and information about the number and type of carers were not available.Items and their distributional characteristics were reviewed. See Table Examination of any floor or ceiling effects on the strain domain demonstrated no outliers. Item-to-item correlations appeared acceptable, as did item-to-total correlations, all generally above 0.30.With the number of factors unspecified, no clear factor pattern was discernible. Next, a six-factor solution for both dimensions (strain and attitude) was examined, based on the suitability of six factors for the original instrument . ExaminaBased on overall item analysis results, including floor and ceiling effects, item-to-item and item-to-total correlation results, and a review of factor analysis results, four poorly-performing items were deleted from the attitude domain: item 5 tries to influence others in order to maintain control of his/her own life; item 12 submissive/gives in to everything done to him/her; item 19 has little control over his/her difficult behavior; and item 27 has patterns of behavior you can foresee. The removal of these items resulted in three distinct subscales for the attitude domain, demonstrating good approximation of simple structure. The three factors, all with variable loadings of 0.30 or above were "Attention-seeking" , "Autonomy" , and "Difficulty" (e.g. "is unpredictable" and "is friendly"). Items were reverse-coded as appropriate.To ensure inter-domain consistency, we removed the same four items from the strain dimension, thus resulting in an acceptable five-factor solution for this domain. The five factors, all with variable loadings of 0.30 or above, are "Affect" , "Job satisfaction" , "Neediness" , "Predictability" , and "Self-direction" . See Tables Following the removal of these 4 items, item-to-subscale correlations were examined for all subscales. In all cases, items correlated moderately to highly with the subscales to which they were assigned via the factor analysis. See Tables The internal consistency reliability  for the attitude and strain total scores was good – 0.79 and 0.95, respectively. Internal consistency reliability for subscales in both domains was excellent in general. See Table Table Table Table Table The M-NCAS demonstrated good psychometric properties based on an analysis of baseline data collected during a clinical trial of risperidone versus placebo, as reported in this preliminary investigation. Item performance, particularly floor and ceiling effects , suggested that four items on the attitude domain were performing poorly from a psychometric standpoint. Two items performed poorly on item correlations as well as factor analysis, while item 12 had a high floor effect as well as poor correlations. The remaining 28 items still capture sufficiently comprehensive data on the domains of interest, based on content review and on the empirical performance of subscales derived from them. The collection of additional data would enhance the confidence of conclusions regarding M-NCAS performance.In general, the M-NCAS demonstrated excellent internal consistency reliability, with only the autonomy subscale producing a Cronbach's alpha below 0.70. Cronbach's alpha values of 0.70 or greater are considered suitable for use in the analyses of group comparisons. Supporting construct validity of the M-NCAS were the moderate and significant correlations to the CMAI and BEHAVE-AD. Correlation with these measures was not expected to be high, given the more expansive focus of the M-NCAS relative to the CMAI and BEHAVE-AD.These results support the use of the M-NCAS for the collection of valid, reliable, and comprehensive information on the burden experienced by nurse carers when caring for dementia patients in long-term care settings. The instrument can assist in staff management by identifying nurse carers with the greatest levels of burden – ideally so that remedial action could be taken, either through extra support for staff members or through the shifting of caseloads from particularly difficult patients for nurses at risk for attrition.There are several limitations to this study. Of major concern is the lack of information on the nurse carers themselves. No data was available as to the type of carer, the duration they had cared for the patient, and their sociodemographic characteristics. The inability to characterize the respondents to the M-NCAS is a limitation in interpreting generalizability of results. No control was attempted for nurse raters, so the possibility exists that measurement properties of the M-NCAS differed across nurse raters – therefore skewing the results. The extent to which rater effects limit the validity of results is likely to be minimal, however, given that multiple subjects were being rated by each nurse rater. Future examination of between-subject properties would be helpful. The sample size for the study is small, relative to the standard recommendations regarding the number of subjects per item for factor analysis. However, we set criteria for relatively moderate to high factor loadings. Of note is that the final recommendations for the measure length are closer to that guideline. Because this was a post-hoc analysis of clinical trial data, the instruments selected for use in assessing construct validity may not have been optimal, in that they did not assess nursing strain per se. However, they did assess the propensity of exhibiting certain behaviors – as does the M-NCAS. Additional data on stability across time (test-retest reliability) is desirable for a full psychometric description of the M-NCAS, and was not available from this study. Finally, this sample of nurse carers was almost exclusively Caucasian. Therefore, the generalizability of these results to nurse carers of different ethnicities, and in different locales is limited.The M-NCAS enables the detection of the presence or absence of specific behaviors similar to checklists , but extends that information by providing a rating of the degree of burden of each aspect rated. It possesses good psychometric properties for use with nurse carers working with Alzheimer's patients in long-term care facilities. The M-NCAS provides a unique approach to identifying both positive and negative behaviors, and to quantifying the amount of stress felt by carers as a result of these behaviors. The nursing staff perspective on residents with dementia is unique , and theBehavioral Pathology in Alzheimer's Disease Rating Scale (BEHAVE-AD)Cohen-Mansfield Agitation Inventory (CMAI)Exploratory Factor Analysis (EFA)Mini-Mental Status Exam (MMSE)Modified Strain in Nursing Care Assessment Scale (M-NCAS)Strain in Nursing Care Assessment Scale (NCAS)LK participated in the data analysis design, data interpretation, and drafting of the manuscript. LF participated in the design, data interpretation, and drafting of the manuscript. GC performed the statistical analyses. MR conceived of the study and participated in data interpretation. HB conceived of the study and participated in both data collection and interpretation. All authors read and approved the final manuscript.


Geohelminth infection is a major health problem of children from rural areas of developing countries. In an attempt to reduce this burden, the Department of Health of the province of KwaZulu-Natal (KZN) established in 1998 a programme for helminth control that aimed at regularly treating primary school children for schistosomiasis and intestinal helminths. This article describes the baseline situation and the effect of treatment on geohelminth infection in a rural part of the province.Ascaris lumbricoides, and Trichuris trichiura, treated twice with 400 mg albendazole and re-examined several times over one year after the first treatment in order to assess the impact of treatment and patterns of infection and re-infection.Grade 3 schoolchildren from Maputaland in northern KZN were examined for infections with hookworm, T. trichiura and especially A. lumbricoides prevalences  were much lower than elsewhere on the KZN coastal plain. Single dose treatment with albendazole was very effective against hookworm and A. lumbricoides with cure rates (CR) of 78.8 and 96.4% and egg reduction rates (ERR) of 93.2 and 97.7%, respectively. It was exceptionally ineffective against T. trichiura . Re-infection with hookworm and A. lumbricoides over 29 weeks after treatment was considerable but still well below pre-treatment levels.The hookworm prevalence in the study population (83.2%) was considerably higher than in other parts of the province whereas T. trichiura infection however demands consideration of alternative treatment approaches.High geohelminth prevalences and re-infection rates in the study population confirm the need for regular treatment of primary school children in the area. The low effectiveness of single course albendazole treatment against  Geohelminth infections are among the most prevalent diseases in developing countries. Children are the group with the highest prevalences and infection intensities and are also very vulnerable to the effects of worm infection. These include nutritional deficiencies ,2 and imConsequently in 1998 the KwaZulu-Natal (KZN) Department of Health initiated a pilot helminth control programme which aimed to regularly apply treatment for intestinal helminths and schistosomiasis to primary school children . This grOur objectives were to describe the initial pattern of geohelminth infection, assess the impact of treatment with 400 mg albendazole and to monitor re-infection after treatment in order to develop recommendations for further local and regional control efforts.The study was conducted in central Ingwavuma district in northern KwaZulu-Natal Figure . This arThe study population was limited to one grade in order to keep disturbance of the school routine low. Grade 3 was selected because it should represent the infection situation in a primary school relatively well . All pup®, SmithKline Beecham). Treatment in the studied schools was administered in April and in October 1998. The study team assisted the nurses with treatment and also recorded those of the study population who were treated and those who were not. During the first round of treatment the pupils were also treated for schistosomiasis with 40 mg/kg praziquantel . However, results regarding schistosomiasis will be reported elsewhere. After the end of the study the participants were included in the normal treatment routine of the control programme.Treatment in all primary schools in the entire district was carried out by school nursing teams from the two local hospitals as part of the helminth control programme. Consenting children from all grades were treated for intestinal helminths with 400 mg albendazole  were nevertheless included in the analysis in order not to increase bias.A. lumbricoides and T. trichiura eggs within one day after preparation [Specimens were kept cool until preparation of the slides. Duplicate 50 mg Kato-Katz cellophane thick smears were prepared from each faecal sample. They were examined by trained microscopists twice: first for hookworm eggs within one hour of preparation and later for paration . AccuratDiarrhoeal specimens and slides that were too dark were not examined. Instead the respective pupils were asked to provide another specimen. Infection intensities are expressed as eggs per gram of faeces (EPG) calculated as the arithmetic mean number of eggs per thick smear multiplied by 20.Data were double entered into Microsoft Excel 97 and corrected for data entry errors. Data analysis was carried out in SPSS 10.0.5 for Windows.Prevalence ratios (PR) and their 95% confidence intervals were calculated using the SPSS "Tables" procedure . Cure raOnly data from the first of the two samples we received per pupil in each post-treatment survey were used for the above calculations in order to make them comparable to the baseline survey. Otherwise differences in sensitivity between surveys would have caused misleading results. Re-infection was however calculated for both obtained samples because the main purpose was not to compare with the baseline situation but to estimate trends in re-infection reliably.A. lumbricoides infection. With a PR of 0.71, males were about 30% less likely to be Ascaris infected than females and they also had lower infection intensities. The prevalence and intensity of hookworm infection were slightly  higher in male pupils and the prevalence of T. trichiura was nearly identical in the two groups.The cumulative prevalence of all three geohelminths combined was as follows: At baseline 90.0% of the pupils were infected with one or more species, 55.9% were infected with two or more species and 30.9% of the children were infected with all three geohelminths. Figure A. lumbricoides infection  was about 27% which is still about 2% higher then the ERR that we found in this study.In contrast, the CR and ERR for T. trichiura: 6 months after treatment the treated pupils had the same prevalence (61%) as a placebo treated control group [A study that was conducted in Durban only about 350 km south of the study area found that albendazole reduced the median EPG of children infected before treatment by 44.1% . The samol group .T. trichiura infection warrant attention. It is unlikely that the low CR and ERR in our study are due to low compliance: Tablet intake was monitored by the school nurses and the study team and only pupils recorded as treated were included in the analysis. Moreover, if a considerable number of children had only pretended to take the tablets, this would also have affected the impact of treatment on hookworm and A. lumbricoides infection. It is also unlikely that the low efficacy is due to drug resistance because of prior treatment. There were no previous control programmes in the area, to our knowledge hospitals and clinics had not been using albendazole or any other benzimidazole until recently and benzimidazoles were also not available in local shops.Although the CR and ERR as measured in most field studies including ours are notoriously unreliable and can only approximately indicate the true treatment success , the verT. trichiura was only 10.5% [High infection intensities, as they are frequently found in schoolchildren, are often mentioned in the literature as a reason for low cure rates ,26 and tly 10.5% . But evely 10.5%  but abouT. trichiura infection as single dose treatments, but other studies show that two or three repeated doses of albendazole on consecutive days are more effective than a single dose [Currently there are no drugs available that are highly effective against gle dose ,26,28,29gle dose .The proportion of children treated in our study can not be regarded as representative for the control programme in general, because study participants were more informed about it than their schoolmates. Therefore it was not analysed statistically. However, it should be noted that although only seven pupils openly refused to be treated, absenteeism was unusually high during the first round of treatment. This improved greatly in the second treatment, when – according to the opinion of school staff – pupils and their parents had realised that treatment was beneficial.A. lumbricoides is also in accordance with the pre-treatment situation.In accordance with the pre-treatment infection levels, hookworm re-infection over 29 weeks after the second treatment was relatively high and thus confirms the need for regular treatment. The moderate re-infection with A. lumbricoides is not too surprising. As opposed to hookworm that is restricted to the humid tropics and subtropics, this helminth also occurs in temperate climates [The markedly higher re-infection with hookworm during the 18 weeks after the second treatment when compared to the 16 weeks after the first treatment is most likely only partly due to the slightly longer re-infection period. The difference in season could also be important ,31: The climates . Its eggT. trichiura complicates conclusions, the data seem to indicate that re-infection was low. Therefore, if a more effective treatment approach could be utilised, the resulting decrease in prevalence and intensity of T. trichiura infections might be relatively long-lasting.Although the low efficacy of treatment for This study has shown that the high prevalence and intensity of geohelminth infection necessitate regular treatment of primary schoolchildren in the study area according to WHO criteria . This isBecause high prevalences in primary schoolchildren may also indicate relatively high community prevalences  it shoulA. lumbricoides infection, but that it is much less effective for the treatment of T. trichiura infection in the study area. It would be important to find out whether albendazole treatment is similarly ineffective against T. trichiura in other parts of the province and to test the effectiveness of treatment alternatives.Our study has also demonstrated that a single course of treatment with 400 mg of albendazole is a powerful tool to control hookworm and To answer the question whether hookworm transmission is really seasonal as this study seems to suggest would necessitate a cohort study over at least one year. Nevertheless it might be worthwhile to investigate when in the year transmission is highest in order to schedule treatment accordingly.None declared.ES conceived of the study and designed it with input from all authors. He conducted the field work with contributions from the other authors, did the statistical analysis and drafted the manuscript. All authors contributed to the final version of the manuscript and read and approved it.The pre-publication history for this paper can be accessed here:


Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of “acquired” resistance.EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.In patients with tumors bearing gefitinib- or erlotinib-sensitive  A specific secondary mutation in the kinase domain of the epidermal growth factor receptor can render cells insensitive to the two kinase inhibitors. This mutation was found in resistant tumors from three of six patients studied KRAS mutations have been associated with some cases of primary resistance to gefitinib or erlotinib [Somatic gain-of-function mutations in exons encoding the epidermal growth factor receptor (EGFR) tyrosine kinase domain are found in about 10% of non-small cell lung cancers (NSCLCs) from the United States ,3, with rlotinib , mechaniAcquired resistance to kinase-targeted anticancer therapy has been most extensively studied with imatinib, an inhibitor of the aberrant BCR-ABL kinase, in chronic myelogenous leukemia (CML). Mutations in the ABL kinase domain are found in 50%–90% of patients with secondary resistance to the drug (reviewed in ). Such mAlthough imatinib inhibits different kinases in various diseases  (reviewed in ), some tEGFR exons 18 to 24 in tumors from five patients who initially responded but subsequently progressed while on these drugs. These exons were also assessed in tumor cells from a sixth patient whose disease rapidly recurred while on gefitinib therapy after complete gross tumor resection. Because of the association of KRAS mutations with primary resistance to gefitinib and erlotinib [KRAS in tumor cells from these six patients. In an effort to explain the selective advantage of cells with a newly identified “resistance” mutation in EGFR—a T790M amino acid substitution—we further characterized the drug sensitivity of putatively resistant EGFR mutants versus wild-type or drug-sensitive EGFR mutants, using both a NSCLC cell line fortuitously found to contain the T790M mutation and lysates from cells transiently transfected with wild-type and mutant EGFR cDNAs.To determine whether lung cancers that acquire clinical resistance to either gefitinib or erlotinib display additional mutations in the EGFR kinase domain, we have examined the status of rlotinib , we alsoTumor specimens, including paraffin blocks, fine needle biopsies, and pleural effusions, were obtained through protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (protocol 92–055  and protEGFR (exons 18–24) and KRAS2 (exon 2) analyses were as published [Genomic DNA was extracted from tumor specimens, and primers for ublished ,7. All sEGFR wild-type) for calibration, this assay detects the presence of the T790M mutation when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (data not shown).A specific exon 20 mutation (T790M) was also detected by length analysis of fluorescently labeled (FAM) PCR products on a capillary electrophoresis device , based on a new NlaIII restriction site created by the T790M mutation (2369 C→T), using the following primers: EGFR Ex20F, 5′-FAM-CTCCCTCCAGGAAGCCTACGTGAT-3′ and EGFR Ex20R 5′-TTTGCGATCTGCACACACCA-3′. Using serially mixed dilutions of DNA from NSCLC cell lines , as per manufacturer's instructions. Minipreps of DNA from individual clones were sequenced using the T7 priming site of the cloning vector.The following primers were used to generate EGFR cDNAs using a QuikChange Site-Directed Mutagenesis Kit  and cloned into expression vectors as described [Two numbering systems are used for EGFR. The first denotes the initiating methionine in the signal sequence as amino acid −24. The second, used here, denotes the methionine as amino acid +1. Commercial suppliers of antibodies, such as the Y1068-specific anti-phospho-EGFR, use the first nomenclature. To be consistent, we consider Y1068 as Y1092. Likewise, the T790M mutation reported here has also been called T766M. Mutations were introduced into full-length wild-type and mutant escribed . The folescribed . All mutescribed . Cells wSee 2. For viability studies, cells were seeded in complete growth medium in black 96-well clear bottom ViewPlates  at a density of 5,000 (H1975 and H2030) or 7,500 cells per well (H3255). Following overnight incubation, cells were grown for 24 h in the supplemented RPMI-1640 medium with 0.1% serum. Cells (in supplemented RPMI-1640 medium containing 0.1% serum) were then incubated for 48 h in the continued presence of gefitinib or erlotinib.The NSCLC cell lines H1650, H1975, H2030, H2347, H2444, H358, and H1734 were purchased from American Type Culture Collection . H3255 was a gift of B. Johnson and P. Janne. Cells were grown in complete growth medium  supplemented with 10% fetal calf serum, 10 units/ml penicillin, and 10 μg/ml streptomycin) at 37 °C and 5% COCell viability was assayed using Calcein AM . Following incubation with gefitinib or erlotinib, monolayers were washed twice with PBS  and incubated with 7.5 μmol Calcein AM in supplemented RPMI-1640 (no serum) for 30 min. Labeling medium was removed, and cells were washed three times with PBS. Calcein fluorescence  was detected immediately using a Victor V multi-label plate reader (PerkinElmer). Three independent experiments were performed for each cell line; each experiment included four to eight replicates per condition.EGFR mutations in three of six individuals whose disease progressed on either gefitinib or erlotinib  initially presented with bilateral diffuse chest opacities and a right-sided pleural effusion. Transbronchial biopsy revealed adenocarcinoma. Disease progressed on two cycles of systemic chemotherapy, after which gefitinib, 250 mg daily, was started. Comparison of chest radiographs obtained prior to starting gefitinib .This 55-y-old woman with a nine pack-year history of smoking underwent two surgical resections within 2 y (right lower and left upper lobectomies) for bronchioloalveolar carcinoma with focal invasion. Two years later, her disease recurred with bilateral pulmonary nodules and further progressed on systemic chemotherapy. Thereafter, the patient began erlotinib, 150 mg daily. A baseline CT scan of the chest demonstrated innumerable bilateral nodules .This 55-y-old female “never smoker” was treated for nearly 4.5 y with weekly paclitaxel and trastuzumab  for adenEGFR mutations, by direct DNA sequencing of exons 19 and 21 [EGFR mutation by direct sequencing .All three specimens from patient 2, including the original lung tumor and the two metastatic samples from bone and lung, showed an exon 19 deletion involving elimination of 11 nucleotides (2238–2248) and insertion of two nucleotides, G and C . These nBoth of the available tumor samples from patient 3 contained a deletion of 15 nucleotides (2236–2250) in exon 19  encoding the EGFR catalytic region in the available tumor specimens.To determine whether additional mutations in the EGFR sequence . However, we did not find any additional mutations in exons 18 to 24 of EGFR, including the C→T change at position 2369 (data not shown). These results imply that alternative mechanisms of acquired drug resistance exist.In three additional patients (case histories not described here) with lung adenocarcinomas who improved but subsequently progressed on therapy with either gefitinib or erlotinib, we examined DNA from tumor specimens obtained during disease progression. In all three patients, we found KRAS2 occur in about one-fourth of NSCLCs. Such mutations rarely, if ever, accompany EGFR mutations and are associated with primary resistance to gefitinib or erlotinib [KRAS mutations confer acquired resistance to these drugs, we performed mutational profiling of KRAS2 exon 2 from tumor specimens from patients 1 to 3, as well as the three additional patients lacking evidence of the T790M mutation. None of the specimens contained any changes in KRAS  and KRAS exon 2 in eight established NSCLC lines .In our own analysis of H1975 (exons 18 to 24), the mutant 2369 T peak resulting in the T790M amino acid substitution was dominant, suggesting an increase in copy number of the mutant allele in comparison to the wild-type allele. The ratio of mutant to wild-type peaks was similar to that of the mutant 2573 G (corresponding to the L858R amino acid substitution) to wild-type T peaks  generated by the specific missense mutation. After PCR amplification with exon-20-specific primers spanning nucleotide 2369, wild-type sequence contains specific NlaIII sites, which upon digestion yield a 106-bp product A. PresenEGFR;We first used DNA from the H1975 cell line (which contains both T790M and L858R mutations) to confirm the specificity of the PCR-RFLP assay. As expected, analysis of these cells produced both the 97- and 106-bp fragments. By contrast, analysis of DNA from H2030 (which contains wild-type EGFR exons 18 to 24 by direct sequencing (data not shown).We next used this PCR-RFLP assay to assess various patient samples for the presence of the specific 2369 C→T mutation corresponding to the T790M amino acid substitution. DNA from the progressing bone and lung lesions in patient 1 produced both the 97- and 106-bp fragments, but DNA from the original lung tumor did not B. The raEGFR cDNAs that encoded the exon 21 and 19 mutations found in patients 1 and 2, respectively. Corresponding proteins  were then produced by transient transfection with expression vectors in 293T cells, which have very low levels of endogenous EGFR [To determine how the T790M mutation would affect EGFR proteins already containing mutations associated with sensitivity to EGFR tyrosine kinase inhibitors, we introduced the specific mutation into Gefitinib inhibited the activity of wild-type and L858R EGFRs progressively with increasing concentrations of drug, as demonstrated by a reduction of tyrosine-phosphorylated proteins A and a dEGFR TK domain mutations (50 of about 0.01 μmol (50 of about 1 μmol (EGFR (exons 18 to 24) and mutant KRAS  appears to be extremely rare. We have not identified this mutation in 155 tumors (see above), and among nearly 1,300 lung cancers in which analysis of erformed ,5,6, onl. However, based upon analogous studies in CML, it is also possible that NSCLC subclones bearing this secondary mutation pre-exist within the primary tumor clone in individual patients, albeit at low frequency [How tumor cells bearing the T790M mutation emerge within gefitinib- or erlotinib-treated patients is a matter of investigation. Subclones bearing this mutation could arise de novo during treatmentrequency . In eithFrom analysis of the crystal structure of the EGFR kinase domain bound to erlotinib, it is has been shown that the wild-type threonine residue at position 790 is located in the hydrophobic ATP-binding pocket of the catalytic region, where it forms a critical hydrogen bond with the drug . The relThe T790M mutation could also affect the kinase activity or alter the substrate specificity of mutant EGFRs, such that a proliferative advantage would be conferred upon cells bearing the mutation. Consistent with this, the H1975 NSCLC cell line reported here to contain both T790M and L858R did not to our knowledge undergo any prior treatment with gefitinib or erlotinib; the doubly mutated cells must have become dominant over time through multiple passages in vitro. This scenario could explain the seemingly contradictory report by others who found the H1975 cell line to be highly sensitive to gefitinib ; our H19Recently, new small-molecule inhibitors have been identified that retain activity against the majority of imatinib-resistant BCR-ABL mutants. The new drugs bind to ABL in an “open” conformation, as opposed to imatinib, which binds ABL in a “closed” conformation ,13. AnalIn some of the patient specimens analyzed, the actual sequencing peaks demonstrating the T790M mutation were smaller than originally anticipated. These results differ from those of acquired resistance mutation in CML , GIST 1,27, and EGFR, either within or outside the tyrosine kinase domain, are likely to exist. It is also possible that EGFR amplification itself plays a role in acquired resistance, since imatinib-resistant clones have been shown to lack resistance mutations but contain amplified copies of BCR-ABL [Since tumor specimens from three additional patients with acquired resistance to EGFR tyrosine kinase inhibitors did not demonstrate the T790M mutation, this specific lesion does not account for all mechanisms of acquired resistance to gefitinib or erlotinib. Given the paradigm established with imatinib, other drug-resistance mutations in  BCR-ABL ,28. NoneFigure S1(A) Patient 1. Serial chest radiographs from before (day 0) and during gefitinib treatment (14 d and 9 mo), demonstrating initial response and subsequent progression.(B) Patient 2. Serial CT studies of the chest before (day 0) and during erlotinib treatment (4 mo and 25 mo), demonstrating initial response and subsequent progression.(C) Patient 3. Serial chest radiographs before (day 0) and during adjuvant gefitinib treatment (3 mo), following complete resection of grossly visible disease. The left-sided pleural effusion seen at 3 mo recurred 4 mo later, at which time fluid was collected for molecular analysis.(951 KB PPT).Click here for additional data file.Figure S2EGFR exon 21 in tumor specimens from patient 1. DNA from the growing lung lesion and the pleural effusion demonstrated a heterozygous T→G mutation at position 2573, leading to the common L858R amino acid substitution.(A) Status of (B) All three specimens from patient 2 showed the same heterozygous exon 19 deletion, removing residues 747–749 and changing the alanine at position 750 to proline.(104 KB PPT).Click here for additional data file.Figure S3See legend for (153 KB PPT).Click here for additional data file.Protocol S1(566 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) accession number for the KRAS2 sequence discussed in this paper is 3845; the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the KRAS2 sequence discussed in this paper is NT_009714.16. Reference EGFR sequence was obtained from LocusLink accession number 1956 and GenBank accession number NT_033968.The LocusLink  and erlotinib , have been developed to inhibit activated EGFR, and studies have shown that they can shrink tumors in some patients. Most patients who respond to these drugs have tumors that carry an alteration (or mutation) in the EGFR gene, which somehow makes their tumors responsive to the drugs.In those patients in whom the drugs work, the tumors shrink initially, but after a while they stop responding and the cancer comes back. The cancer has, as researchers describe it, become resistant to the drugs. Understanding how tumors become resistant is important to develop new and better drugs.They asked patients who initially responded to erlotinib or gefitinib but then became resistant to consent to studies allowing further analysis of tumor tissue during and after drug treatment. They then re-examined the EGFR gene in these tumor samples.They found that tumors from all patients carried mutations in the EGFR gene that are known to make them responsive to the drugs. In addition, three of the post-treatment tumors had an identical second mutation in their EGFR gene. Biochemical studies showed that these secondary alterations made the original drug-sensitive EGFR less sensitive to drug treatment. The numbers are small but suggest that this secondary resistance mutation could be quite common. Tumor cells from the three other patients didn't have this mutation, which suggests that there are other ways for lung cancers to become resistant to gefitinib and erlotinib.Larger studies are needed to confirm that this particular mutation is a major cause of resistance against the two drugs. It is also important to find out what causes resistance in the other cases. And knowing about this resistance mutation will help researchers to develop drugs that will work even against tumors with the mutation.The following pages contain some information on the EGFR kinase inhibitors.http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htmU. S. Food and Drug Administration information page on Iressa (gefitinib): http://www.cancerhelp.org.uk/help/default.asp?page=10296Cancer Research UK information page about erlotinib (Tarceva):


We often think of ourselves as either a day person or a night person—one who rises with the sun, raring to go, or one who prefers to stay up through the night to get things done. Regardless, we each have our regular waking and sleeping cycles. It's been known for some time that variations in sleep and wakefulness are part of our circadian rhythm, or molecular clock. A portion of the brain called the hypothalamic suprachiasmatic nucleus (SCN) regulates this biorhythm. When this area of the hypothalamus is destroyed in animal models, the circadian rhythm is disrupted. Two transcription factors (proteins that regulate gene expression) called Bmal1 and Clock regulate aspects of circadian rhythm, possibly by regulating neurons in the SCN.Other aspects of human physiology are also regulated in a circadian manner. Besides altering sleep and wakefulness patterns, ablation of the SCN alters the ability to regulate sugar levels. Sugar (glucose) levels must be maintained within fairly narrow limits for survival. This regulation is controlled in part by a balance between blood sugar level and insulin production (insulin lowers the blood sugar level). In people and in mouse models, both glucose level and insulin level are subject to circadian rhythms. It isn't clear, however, if this is a behavioral effect, whereby the disruption of the SCN might alter our feeling of being well fed—that is, being sated—as eating has a profound effect on blood sugar levels.Garret FitzGerald and colleagues tested the effect of the molecular clock genes in glucose regulation (homeostasis) by examining mice in which Clock and Bmal1 were impaired. In normal mice they observed a peak in glucose levels early in the day. This diurnal regulation was lost in the mutant mice. Furthermore, whereas the normal mice could fairly easily return their glucose levels to normal when they were artificially treated with insulin, this ability was severely impaired in the mutant mice. What's more, a high-fat diet amplified this circadian variation in the normal animals, but the rhythm was abolished in the mutants on a high-fat diet. Thus, the authors demonstrated that circadian control of blood glucose levels is due directly to the presence of these transcriptional factors rather than due to some other behavioral effect that ablation of the hypothalamus might have caused. It's possible, therefore, that besides what we eat, our internal circadian clock could also be an important regulator of blood sugar levels.What is still left to be explored is whether the change in glucose that results from disruption of the Clock and Bmal1 genes is due to the transcription factors' effect as circadian regulators or to an activity of these transcription factors that is unrelated to circadian rhythm generation. But the study does raise the possibility that when you eat may be as important to your health as what you are eating.


Anopheles gambiae populations is important for the current tactics in mosquito control and for the proposed strategy using genetically-modified mosquitoes (GMM). Genetically-isolated mosquito populations on islands are a potential site for testing GMM. The objective of this study was to determine the genetic structure of A. gambiae populations on the islands in Lake Victoria, western Kenya.Understanding the genetic structure of island A. gambiae populations from five islands on Lake Victoria and six villages from the surrounding mainland area in the Suba District were examined using six microsatellite markers. The distance range of sampling sites varied between 2.5 and 35.1 km.The genetic diversity and the population genetic structures of 13 ST = 0.019) and between the island and mainland populations (FST = 0.003). A total of 12 private alleles were found, and nine of them were from the island populations.A similar level of genetic diversity between island mosquito populations and adjacent mainland populations was found. The average number of alleles per locus was 7.3 for the island populations and 6.8 for the mainland populations. The average observed heterozygosity was 0.32 and 0.28 for the island and mainland populations, respectively. A low but statistically significant genetic structure was detected among the island populations (FA level of genetic differentiation between the island and mainland populations was found. Large extent of gene flow between the island and mainland mosquito populations may result from wind- or human-assisted dispersal. Should the islands on Lake Victoria be used as a trial site for the release program of GMM, mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored. Plasmodium [Despite 50 years of malaria vector control efforts, malaria remains a major public health threat in tropical and subtropical countries -3. In reasmodium  has signasmodium .A. gambiae cell lines were successfully transformed with the Hermes element [Minos transposable element bearing an exogenous gene was efficiently integrated into the genome of Anopheles stephensi [A. gambiae [A. gambiae genome sequences will greatly facilitate identification and cloning of parasite-inhibiting genes [One potential alternative malaria control strategy is based on the genetic disruption of mosquito vector competence -13. This element ,15, and tephensi ,17. Gene gambiae , and gen gambiae . Availabng genes .A. gambiae population genetic structure in the African continent has been examined [A. gambiae; however, no significant genetic structure was detected for mosquito populations between western Kenya and West Africa. The minimum area associated for a deme of A. gambiae in western or coastal Kenya is larger than 50 km [et al. [Anopheles arabiensis populations from the high plateau of Madagascar and those from Réunion and Mauritius islands (FST ranges from 0.080 to 0.215). Population substructure was also detected on the island of São Tomé, West Africa [The success of the transgenic mosquito approach depends on the spread and even fixation of parasite-inhibiting genes into natural populations. Presently, releasing transgenic mosquitoes to the field is premature. Isolated islands have been suggested as an ideal natural site for testing transgenic mosquito release strategies and spatial spreading of transgenes ,21,22. Iexamined -28. Thesan 50 km . Simard  [et al.  found a A. gambiae mosquitoes from five islands on Lake Victoria and the surrounding mainland in western Kenya. This information is valuable for selecting field sites to test transgene release strategies and evaluating the spread of transgenes in nature.The present study examined the genetic diversity and the population genetic structures of A. gambiae mosquitoes are the major malaria vectors in this region [Anopheline female mosquitoes were collected from seven villages on five islands in Lake Victoria and from six villages in the mainland Suba District, western Kenya Fig. . The sams region .A. gambiae sensu lato (s.l.) specimens were separated from other anophelines according to the identification key provided by Gillies and Coetzee [At least 170 anopheline mosquitoes were collected from four to 28 houses within each village using the pyrethrum spray collection method . Mosquit Coetzee  and thenA. gambiae species complex cannot be identified by morphology alone [A. gambiae s.l. females per village were tested. If the initial PCR testing failed to amplify for a sample, then the PCR analysis was repeated once or twice until successful amplification was achieved. If a sample could not be identified after three PCR amplifications, it was scored as unknown.PCR analysis was conducted for species identification using the rDNA-PCR method because individual species within the gy alone . About 1A. gambiae populations from the Lake Victoria islands and the surrounding mainland in western Kenya).Six microsatellite markers were used for specimen genotyping, including AGXH1D1 and AGXH131 of Chromosome X, AG2H46 and AG2H79 of Chromosome 2, and AG3H29C and AG3H33C of Chromosome 3 ,18,22,232, 1.0 μg BSA, 1.0 unit Taq polymerase and about 20 ng genomic DNA. Cycling conditions in a MJ Research PTC-220 thermocycler were 35–40 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 45 seconds. Allele sizes were determined using Gene ImagIR computer software [A Li-Cor Model 4200 Automated DNA Analyzer  was used for gel electrophoresis. For the apparatus to detect PCR products, one primer in every pair of microsatellite primers must be fluorescently labelled. To reduce the cost associated with synthesis of fluorescently labelled primers, we used the "tailed primer" method ,35; thatsoftware . The allIS statistics and probability test were used to determine whether distortion from HWE resulted from heterozygosity deficiency or excess using. Because the probability test is robust to low allele frequencies, rare alleles were not pooled. Variations in heterozygosity among the populations were analyzed following Weir's method [ST) using FSTAT 2.8 [ST statistic appears to be more sensitive to detect intraspecific differentiation than RST [Microsatellite polymorphism was measured by the number of alleles and heterozygosity at each locus. Using the probability test available in the GENEPOP computer program , conforms method , using tSTAT 2.8 . FST stathan RST ,41. The than RST  were calthan RST . A dendrST/(1 - FST) against the natural logarithm of straight-line geographical distance between population pairs [The isolation-by-distance model of population genetic structure was tested by linear regression of pair-wise Fon pairs . Statiston pairs .A moderate to high level of polymorphism was found in six loci across the 13 populations See the . The thrA. gambiae populations [A total of 14.1% loci (11 out of 78 tests) showed significant departure from Hardy-Weinberg equilibrium, all due to heterozygote deficiency. This was caused entirely by heterozygote deficiency in the locus AG2H46, a locus known for the presence of null alleles in western Kenyan ST = 0.019, P < 0.001) was almost twice as high as the six mainland populations . Genetic differentiation between island and mainland populations was also small . Pair-wise comparisons between all populations revealed that only seven pairs  exhibited significant FST values, and six of them were between an island and a mainland population.A low, but significant, genetic structure was detected among the seven island and the six mainland populations Table . The genST/(1 - FST) (P < 0.001), suggesting that the population genetic structure of A. gambiae populations from the island and mainland is consistent with the isolation-by-distance model. When the Ruri population is removed from the analysis, the correlation was still statistically significant (P = 0.015). Therefore, the population genetic structure of our study populations is consistent with the isolation-by-distance model. The cluster analysis revealed that the Ruri population, located farther inland from the other populations, was out-grouped from other populations with a significant bootstrap value, while other mainland and island populations were intermixed with non-significant branch bootstrap values  and among the six villages in the mainland in an area of approximately 40 × 20 km2 (FST = 0.010). The degree of genetic differentiation between the island populations in this study was less than for the island A. gambiae populations of São Tomé, western Africa (FST = 0.032) [ST estimates in the populations in this study were probably caused by shorter distance between islands (3–15 vs. 23–38 km) [ST estimates for the mainland populations in this study were comparable to other studies on the western Kenya populations (FST = 0.0033) [ST = 0.003). Thus, there is a very small degree of genetic isolation between island and mainland populations. This estimation is consistent with the private allele distribution in the studied populations, in which nine of the 12 private alleles were from the island populations. Further evidence for a small degree of genetic differentiation between island and mainland populations is from pair-wise population comparisons in which six out of the seven pairs that exhibited significant genetic differentiation were between an island population and a mainland population.A small but statistically significant genetic structure was detected for = 0.032) . The low3–38 km)  and a la 0.0033) ,27. The A. gambiae is usually less than 1 km [et al. [A. gambiae mosquitoes was related to the predominant wind direction at night, suggesting that wind assisted the dispersal of mosquitoes from their breeding site. A. gambiae have been shown to fly up to 7 km with the assistance of wind [The low level of genetic differentiation between island and mainland mosquito populations implies large gene flow between the two areas (83.1 migrants per generation). The normal flight range of han 1 km . The dis [et al.  found th of wind ,53. ThisAedes polynesiensis populations from islands found no significant effect of geographic distance on the population genetic structure, but detected a significant correlation between gene flow and commercial traffic by planes and/or boats between islands [A. arabiensis to the Mascarene islands and Madagascar was thought to be caused by human transportation by steamship lines [A. gambiae larvae were collected at the bottom of a wooden fishing boat [Mosquito migration may also be assisted by human activities. A study on  islands . The intip lines ,56. In Ling boat . RushingA. gambiae populations have implications for the ecological safety evaluation of the transgenic mosquito release program. During the initial field test of environmental safety and public health consequences by transgenic mosquito release, ideal sites would be islands that are totally genetically isolated from other islands and the mainland, with a sufficient number of human inhabitants and active malaria transmission on the island. Such an island may be extremely difficult to find, so islands with some genetic isolation from the mainland may have to be chosen. If so, the Lake Victoria islands could be used as field test sites; however, due to potential gene flow between the islands and between the islands and the mainland, mosquito dispersal between the islands and between the islands and the mainland should be vigorously monitored. After the release of the genetically modified mosquitoes, long-term monitoring programs should be launched to evaluate the spread of the transgenes to any unintended areas. In addition, methods to minimize the negative effects of transgene leak need to be developed prior to the field trial of transgene release [The results of this study of the population genetic stricture of island and mainland  release .This study showed that a low level of genetic differentiation existed between the island and mainland populations and no any genetically-isolated population was found among the 13 mosquito populations. If the islands on Lake Victoria were used as a trial site for the program to release genetically-modified mosquitoes, short-term and long-term mosquito dispersal between the islands and between the island and the mainland should be vigorously monitored.HC conducted species identification using PCR, microsatellite analyses and drafting the manuscript. NM was responsible for sample collection, and participated in species identification and drafting the manuscript. JB and GY supervised the study, and assisted data analysis and manuscript preparations.A. gambiae populations from the Lake Victoria islands and the surrounding mainland in western Kenya.A table of sample size, allelic number, heterozygosities and breeding coefficient of 13 Click here for file


Fetal growth restriction is associated with significantly increased risks of neonatal death and morbidity and with susceptibility to hypertension, cardiovascular disease and NIDDM later in life. Human birth weight has a substantial genetic component, with at least a quarter of the variation attributable to additive genetic effects.One hundred twenty-five subjects (83 control and 42 case) were selected using stringent inclusion/exclusion criteria. DNA sequencing was used to identify 26 single nucleotide polymorphisms in the pituitary growth hormone gene (GH1) at which all subjects were genotyped. Association with fetal growth restriction was tested by logistic regression for all sites with minor allele frequencies greater than 5%.Logistic regression identified significant association with fetal growth restriction of C alleles at sites -1 and +3 (relative to the start of transcription) that are in complete linkage disequilibrium. These alleles are present at higher frequency (6% vs. 0.4%) in fetal growth restricted subjects and are associated with an average reduction in birth weight of 152 g in normal birth weight and 97 g in low birth weight subjects.There is suggestive association between fetal growth restriction and the presence of C alleles at sites -1 and +3 of the pituitary growth hormone gene. Fetal growth restriction (FGR) is a major risk factor for illness in the perinatal period and throughout life, with the smallest 7.5 percent of infants accounting for two-thirds of infant deaths . Term, lIt is difficult to distinguish the genetic and environmental components of human birth weight variation, but recent studies support a major genetic component to birth weight variation. Clausson et al.'s  study ofThe five genes of the human growth hormone locus reside within about 45 kb on chromosome 17 . PituitaHere we use DNA sequencing to identify and to determine the frequencies of both 12 newly-identified single nucleotide polymorphisms (SNPs) in the promoter and coding region of the GH1 gene and 15 previously reported SNPs ,18. Usinth percentile of size for gestational age [th percentile of female and lowest 6.5 percentile of male deliveries [DNA was extracted from placental tissue from 125 live births  at Baystate Medical Center  in a case-control study of the genetic predisposition to fetal growth restriction. All subjects were Caucasian, both Hispanic and non-Hispanic. Classification of newborns as fetal growth restricted  followed the definition of the American College of Obstetricians and Gynecologists as those newborns below the 10onal age . Our cutliveries . StringeThe region from -624  to +1,726 (197 nucleotides after the termination codon) of GH1 was amplified in two overlapping fragments: -624 to +541  and +450 to +1,726 . Approximately 50 ng of DNA was used as a template in a polymerase chain reaction with 30 cycles of 95°C (1 minute), 62°C (1 minute), and 72°C (2 minutes 30 seconds). PCR products were purified from agarose using a QIAquick PCR Purification kit (Qiagen) and sequenced directly with BigDye v2.0 chemistry (Applied Biosystems) and run on either an ABI Prism 377 or 3100 automated DNA sequencer. PCR products were sequenced with the PCR primers and additional internal primers: 5' AAGCACAGCCAATAGATTG 3', -459 to -441; 5' GCACAAGCCCGTCAGTGGCC 3', -108 to -89; 5' GGATTTTAGGGGCGCTTACC 3', +71 to +90; 5' CATCTCCCTGCTGCTCATC 3', +931 to+949; 5' GCGCTTGGGYACTGTTCCCT 3', +1280 to +1299.Sequence traces were aligned and assembled into contigs by the program Polyphred . ContigsThe five genes of the human growth hormone locus exhibit high sequence similarity, and the paralogous regions corresponding to the portion of GH1 sequenced in this study  were multiply aligned. Nucleotide positions , as defined in the methods. Of the invariant sites, 5 are polymorphic in GH1, while 21 of the variant sites are polymorphic. A comparison of the proportion of polymorphic sites at invariant and variant sites by Fisher's Exact Test is highly significant (P <<0.001). This result indicates that there is a strong bias for polymorphisms in GH1 to occur at the minority of sites that exhibit sequence divergence among the paralogous genes. The high correspondence between the sequence of minor alleles in GH1 and nucleotides present in paralogs of GH1 supports previous assertions that the unusually high polymorphism of the GH1 gene is driven by gene conversion ,18.A proportion of this bias may be explained by selective constraint, in that sites that are polymorphic within GH1 may be under less selective constraint and thus more free to exhibit sequence divergence among paralogs. However, to entirely explain the bias towards polymorphism at sites of divergence, one must assume that about 1,804 (91%) of the invariant sites are selectively constrained and not free to vary. Given that a substantial proportion  of the sequence surveyed in this study is composed of introns, this assumption seems unreasonable.Logistic regression Table  was perfAlthough the A/C polymorphism at site -1 was not significant in a reduced model, we decided to investigate this site for three reasons. First, the C allele at site -1 is in complete linkage disequilibrium with C at site +3. Second, both of these sites are located at the start of transcription, making them good candidates for affecting the level of transcription of GH1. Third, the only paralog that shares C at these sites is CS-5, a pseudogene, consistent with the possibility that C at sites -1 and +3 is disadvantageous. The C allele at both sites exhibits a much higher frequency in low birth weight (6%) versus normal birth weight 0.4%) subjects. Restricting examination only within the normal or low birth weight subjects, the AC genotype is associated with an average reduction of birth weight of 152 g and 97 g in normal and low birth weight subjects, respectively. However, this difference does not achieve significant in an ANOVA on the A/C polymorphism and gestational age  did among British army recruits (0.4% vs. 0.3%) but that among the low birth weight subjects the frequency of the C allele is substantially higher (6%). Comparing all the other allele frequencies . There are two possible explanations for this observation. First, site -1 could be a hotspot for nucleotide mutation, with transversions from A to C/T occurring often. Second, GH1 may be the recipient of gene conversion events from more than one paralog within the GH locus. Among the four other genes of the GH locus, all three alleles observed at site -1 occur Table . Given tIt must be pointed out that the common wisdom is that GH1 plays no role in regulating fetal growth because the GH receptor is expressed fairly late in gestation and because anencephalic infants or those born without a pituitary achieve nearly normal length . ImportaIn a stringently selected set of subjects, C alleles at sites -1 and +3 relative to the start of transcription of GH1 have a higher frequency (0.4% vs. 6%) in fetal growth restricted newborns. These two alleles are in complete linkage disequilibrium and their presence is associated with a reduction in birth weight of 152 g in term, normal birth weight subjects and 97 g in term, low birth weight  subjects. In combination with environmental, behavioural and other genetic factors, these alleles may contribute to fetal growth restriction.The author(s) declare that they have no competing interests.RMA conceived the project and directed its design and execution. CC and RV performed the molecular genetic work and participated in preliminary analyses. TKB performed the subject selection.The pre-publication history for this paper can be accessed here:


Over 250 million years ago (mya), all the continents of Earth formed a single land mass called Pangaea. Some 50 million years later, this supercontinent began to split in two, forming Laurasia—now North America, Asia, and Europe—and Gondwana—present-day Antarctica, Australia, South America, Africa, and India. After another 50 million years, Gondwana, too, broke up. At the end of the Cretaceous period, New Zealand split off (about 80 mya), then South America and Australia separated from Antarctica (about 35 mya). Fairy-tale quality aside, the story of continental drift fits comfortably with the geological and fossil record and feeds our understanding of current distributions of plant biodiversity.Nothofagus—the southern beech tree, a plant whose 80-million-year-old fossil history goes back to the days of Gondwana—has polarized views on the nature of Southern Hemisphere biogeography.Although we know how and when Pangaea broke apart, the distribution of fossils of the same species on many different continents, separated by vast ocean waters, challenges us to explain how they got there. Plant life on New Zealand, for example, shares striking similarities to that on other Southern Hemisphere land masses, but scientists have yet to agree on how this came to pass. In particular, one genus, Nothofagus seeds are not considered ocean-worthy vessels, many believe vicariance is the only possible explanation.One theory suggests that geographic barriers  would have prevented species expansion after the break-up of the continents, so similar contemporary species must have already existed in both places before New Zealand broke away from Gondwana. In this scenario, called vicariance, ancestors of existing lineages drifted with the repositioned land masses. Another hypothesis, born of existing distributions and fossil data, suggests that long-range oceanic dispersal is more likely. But since Peter Lockhart and colleagues argue that a clear picture of the divergence dates of various southern beech species could help clarify the relative contributions of vicariance versus dispersal. But they would need significant lengths of DNA sequences to reliably characterize the evolutionary history of each species.Nothofagus subgenera—Lophozonia, Fuscospora, and Nothofagus—from South America, Australia, and New Zealand. Reconstructing the trees' evolutionary relationships (phylogeny) based on analyses of their chloroplast sequences, the authors discovered a nuanced evolutionary history that supports vicariance for some species and dispersal for others.Consequently, Lockhart and colleagues analyzed a 7.2-kilobase fragment of the chloroplast genome  for 11 species of three Assuming that beech was present throughout Gondwana (which fossil data support), the sequence of the Gondwana breakup should be reflected in the beech's phylogeny. New Zealand beeches should be more distantly related to both Australian and South American species, because of the greater period of separation—65 million years compared to 30 million years. Yet Australian and New Zealand beeches are more closely related to each other than to South American species, which reflects more recent relationships. Given that fossils of all beech subgenera extend back to the New Zealand Cretaceous period, the dating of splits and the nature of the relationships indicate extinction of beech lineages within current subgenera in New Zealand, and possibly in Australia and South America.Lophozonia and Fuscospora species are too recent to have roots in Gondwana, indicating a role for transoceanic dispersal. The evolutionary relationship between the Australasian and South American Fuscospora lineages, however, is consistent with vicariance. These divergence results, the authors conclude, indicate that current distributions of Nothofagus cannot be explained solely by continental drift (followed by extinction of some species) and that contemporary New Zealand Nothofagus species are not direct descendants of the beeches thought to have reached the island after the split from Antarctica.Lockhart and colleagues' analyses suggest that the relationships of the Australian and New Zealand Nothofagus seeds—and stresses the need to consider more complex hypotheses to explain something as dynamic and complex as the evolutionary history of biodiversity.Taken together, the results highlight the need for caution in evaluating fossil evidence. The fossil record doesn't necessarily capture when a species first appeared, and a continuous fossil presence can mask extinctions and reinvasions. The authors conclude that their molecular data make the case for investigating possible mechanisms of long-range dispersal—especially the dispersal properties of