Nucleotide sequence of the dsRNA genomic segment 7 of Simian 11 rotavirus.

A full length cDNA copy of dsRNA segment seven of Simian 11 rotavirus has been obtained by standard molecular cloning techniques. Segment seven codes for the non-structural viral protein NCVP4 and is 1104 nucleotides in length with putative 5'- and 3'- terminal non-coding regions of 25 and 134 residues respectively. The longest open reading frame of 315 codons extends from nucleotide 26 to 970 inclusive. However, the presence nearby of two other AUG codons makes it unclear which codon is used for initiation. The second AUG conforms to the Kozak consensus sequence and if utilised, would yield a protein 312 amino acids in length with a nett charge at pH7 of -2.5. Determination of the gene 7 sequence indicates that terminal sequence conservation among rotavirus gene segments is limited to three and two nucleotides at the 5' and 3' ends of the plus strand, respectively.     

Exocytosis and growth do not occur only at hyphal tips

Mycologists have put extreme emphasis on hyphal tip growth as the primary mode of growth in filamentous fungi. Much attention has also been focused on the exocytosis of extracellular enzymes from hyphal tips. However, growth and exocytosis commonly occur at hyphal locations other than tips. Here I briefly review our limited understanding of growth and exocytosis during intercalary hyphal extension, subapical branch initiation, septum formation and secondary wall thickening. Secretion of extracellular enzymes and adhesion molecules from subapical hyphal regions is also discussed. Recent research using advanced live-cell imaging techniques (e.g. Hayakawa et al., 2011 in this issue) is providing new insights into the mechanistic basis of many of these processes.     

Accidents in children do not happen at random: Predictable time-of-day incidence of childhood trauma

In a prospective study, 15,110 childhood traumas were recorded by the Pediatric Surgery Service (CHUV, Lausanne) between January 1, 1990 and December 31, 1997. The exact clock hour when the injury occurred and other germane data were obtained. Time series thus obtained were analyzed by several statistical (ANOVA, cosinor, χ², Table Curve, etc.) methods. High statistically significant circadian patterns were detected with a trough at night—almost no traumas/hour (t/h), and a peak in the afternoon (～16:00h)—9.3±0.4 (SD) t/h. Such 24h variation was validated for the whole sample for the entire 8yr study span as well as the data of each year. Neither gender- nor age-related differences in the 24h pattern were detected between children under 5 yr of age, who have not yet attended school and children from 5 to 16 yr of age, who attend school. Small but statistically significant differences in the 24h patterns were observed when categorized by the type of activity associated with the trauma and the place of trauma occurrence. The great stability of the 24h pattern in childhood trauma over the 8yr study span suggests an endogenous origin in addition to the role presumably played by environmental factors. Periods of 12 and 8 h were also detected in the time series. The afternoon peak time of childhood traumas differs from that of adults, which is located ～04:00h in rotating shift workers and automobile drivers and 06:00–08:00h in adult day-workers. The validation of a circadian pattern in childhood traumas with an afternoon peak should be taken into account in the design of children's preventative injury programs.     

Accidents in children do not happen at random: predictable time-of-day incidence of childhood trauma

In a prospective study, 15,110 childhood traumas were recorded by the Pediatric Surgery Service (CHUV, Lausanne) between January 1, 1990 and December 31, 1997. The exact clock hour when the injury occurred and other germane data were obtained. Time series thus obtained were analyzed by several statistical (ANOVA, cosinor, χ², Table Curve, etc.) methods. High statistically significant circadian patterns were detected with a trough at night—almost no traumas/hour (t/h), and a peak in the afternoon (∼16:00h)—9.3±0.4 (SD) t/h. Such 24h variation was validated for the whole sample for the entire 8yr study span as well as the data of each year. Neither gender- nor age-related differences in the 24h pattern were detected between children under 5 yr of age, who have not yet attended school and children from 5 to 16 yr of age, who attend school. Small but statistically significant differences in the 24h patterns were observed when categorized by the type of activity associated with the trauma and the place of trauma occurrence. The great stability of the 24h pattern in childhood trauma over the 8yr study span suggests an endogenous origin in addition to the role presumably played by environmental factors. Periods of 12 and 8 h were also detected in the time series. The afternoon peak time of childhood traumas differs from that of adults, which is located ∼04:00h in rotating shift workers and automobile drivers and 06:00-08:00h in adult day-workers. The validation of a circadian pattern in childhood traumas with an afternoon peak should be taken into account in the design of children's preventative injury programs.     

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.     

Asymptomatic Helicobacter pylori infection and iron deficiency are not associated with decreased growth among Alaska Native children aged 7-11 years

Introduction: Alaska Native children have high Helicobacter pylori infection and iron deficiency prevalences, and their average height‐for‐age is lower than US reference populations. During a clinical trial to determine the impact of H. pylori treatment on iron deficiency, we evaluated the effects of H. pylori infection and treatment on growth. Materials and Methods: We measured height and weight for children aged 7–11 years in western Alaska using village‐based measuring devices. H. pylori infection was determined by urea breath test and iron deficiency using serum ferritin. Children with H. pylori infection and iron deficiency entered the treatment phase and received iron alone or iron plus triple therapy for H. pylori. Follow‐up evaluations occurred at 2, 8, and 14 months. We evaluated the association between baseline H. pylori infection and growth; among children in the treatment phase, we also assessed the effect of H. pylori resolution on growth. Results: At baseline, 566 (87.1%) of 650 children were infected with H. pylori. Neither height and weight, nor body mass index differed by H. pylori infection status. Of 189 children in the treatment phase, 20 (10.6%) were uninfected at all three follow‐up periods, and 54 (28.6%) were uninfected for one or two periods. Compared with continuously infected children, children in these two groups had little evidence of improvements in any of the measured growth outcomes. Conclusions: H. pylori infection is not related to growth among Alaska Native children aged 7–11 years. Growth deficiency should not be considered an indication for H. pylori therapy.     

Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques

SIV(mac239) infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIV(mac239)-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIV(mac239) infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.     

Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo

Olig gene expression is proposed to mark the common progenitors of motoneurons and oligodendrocytes. In an attempt to further dissect the in vivo lineage relationships between motoneurons and oligodendrocytes, we used a conditional cell-ablation approach to kill Olig-expressing cells. Although differentiated motoneurons and oligodendrocytes were eliminated, our ablation study revealed a continuous generation and subsequent death of their precursors. Most remarkably, a normal number of oligodendrocyte precursors are formed at day 12 of mouse development, after all motoneuron precursors have been killed. The data presented herein supports a sequential model in which motoneuron and oligodendrocyte precursors are sequentially generated in vivo from neuroepithelial stem cells, but do not share a common lineage-restricted progenitor.     

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.     

Coding assignment and nucleotide sequence of simian rotavirus SA11 gene segment 10: location of glycosylation sites suggests that the signal peptide is not cleaved.

A cloned DNA copy of simian rotavirus SA11 genomic segment 10 was used to confirm the assignment of the nonstructural glycoprotein NCVP5 to this gene. Determination of the nucleotide sequence for gene 10 indicated that NCVP5 is 175 amino acids in length and has an N-terminal hydrophobic region with the characteristics of a signal sequence for membrane translocation. Unexpectedly, this region was also the location for the only two potential glycosylation sites within the molecule, asparagine residues 8 and 18. The carbohydrates carried by NCVP5 were of the high-mannose type, Man9GlcNAc and Man8GlcNAc, with the mannose 9 species predominating; no complex oligosaccharides were present. If these asparagine residues are the sites for carbohydrate attachment, this implies that cleavage of the putative signal peptide does not occur during the maturation of this nonstructural glycoprotein.     

Hypotension and bradycardia during caloric restriction in mice are independent of salt balance and do not require ANP receptor

We hypothesized that caloric restriction (CR)-induced hypotension would correlate with increased sodium excretion through an atrial natriuretic peptide (ANP)-dependent mechanism. To test this hypothesis, the cardiovascular parameters of c57/Bl mice were measured with radiotelemetry while urine was collected. The 23-h mean blood pressure (BP) dropped from 108.6 +/- 1.8 to 92.7 +/- 2.4 mmHg, and 23-h heart rate dropped from 624 +/- 5 to 426 +/- 13 beats/min over 7 days of CR at 29 degrees C. Contrary to our hypothesis, urine sodium excretion decreased by 55% by day 7 of CR. Consistent with decreased sodium excretion was the drop in plasma ANP (from 82.4 +/- 4.3 to 68.0 +/- 5.8 pg/ml). To explore the possibility that CR lowers BP through an ANP receptor-dependent mechanism that is independent of its effect on sodium retention, we measured the cardiovascular parameters of mice deficient in the ANP receptor (NPR1(-/-)) or the ANP clearance receptor (NPR3(-/-)). Mean BP fell from 117.1 +/- 3.9 to 108.0 +/- 4.7 mmHg in the NPR1(-/-) mice and from 87.0 +/- 2.4 to 78.4 +/- 1.7 mmHg in the NPR3(-/-) mice during CR. These data indicate that the hypotension induced by CR does not depend on increased sodium excretion. Rather, it appears that the mouse responds to the low BP induced by CR with an increase in sodium reabsorption. Furthermore, circulating ANP levels and data from NPR1(-/-) and NPR3(-/-) mice suggest that the ANP pathway may not be involved in the cardiovascular response to CR.     

Sialyl Lewisx epitopes do not occur on acute phase proteins in mice: relationship to the absence of α3-fucosyltransferase in the liver

Mice are frequently used in models for the study of immunological processes related to inflammation. Since it is known that the degree of fucosylation of human acute phase proteins (APPs) is altered as a consequence of an inflammatory response, we have undertaken this study to gain more insight into the fucosylation of acute phase proteins as it occurs in mouse liver. Mice carrying the cluster of the three genes encoding human α1-acid glycoprotein (AGP), one of the well known APPs, were used and the fucosylation of AGP was assessed. A complete absence of fucosylation on the transgenic human AGP was found, which is in sharp contrast to AGP in human serum, of which a major proportion is normally α3-fucosylated. Remarkably, a large proportion of mouse AGP did contain fucose residues. Fucosylation was also detected on another APP, mouse protease inhibitor (PI). α3-Fucosylation of the transgenic human AGP can be achieved in vitro, using an α3/4-fucosyltransferase (α3/4-FucT) isolated from human milk, showing that the glycoprotein is not intrinsically resistant to fucosylation. Upon subsequent measurement of the activities of the possible fucosyltransferases present in liver membranes of parent and transgenic mice, only an N-linked-core α6-FucT and no α2-, α3- or α4-FucT activity was detected. This indicates that fucose residues found on the mouse serum proteins AGP and PI, which are synthesized in the liver, are most probably in α6-linkage to the core chitobiosyl unit. Interestingly, both α6- and α3-FucT activity was detectable in human liver membranes. None of the above mentioned findings were influenced by the induction of an acute phase response by administration of bacterial lipopolysaccharide. This study shows that: (a) α6-FucT is probably a protein specific-glycosyltransferase, since mouse AGP, but not human AGP, may be used as an acceptor; (b) in contrast to human liver, mouse liver does not express any α3-FucT-activity, thereby making the mouse incapable of producing the Sialyl Lewisx epitope on APPs, which is an important part of the inflammatory reaction in humans. This last finding indicates that the mouse is not suitable as a model for the study of those phenomena related to inflammation in humans, in which glycosylation of acute phase proteins could play a significant role. © 1998 Rapid Science Ltd     

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.     

Recurrent genomic rearrangements are not at the fragile sites on chromosomes 3 and 5 in human renal cell carcinomas

Summary It has been suggested that fragile sites on human chromosomes predispose to specific rearrangements seen in cancer. Renal cell carcinoma is characterised by recurrent aberrations of chromosome 3p and frequent rearrangements of chromosome 5q. To investigate whether there might be an association between fragile sites and recurrent breakpoints in renal cell carcinoma, we have determined the breakpoints observed in 50 tumours and compared them to the known fragile sites on chromosomes 3 and 5. No correlation between fragile sites and cancer-related breakpoints in renal cell carcinomas was found.     

In vivo forces generated by finger flexor muscles do not depend on the rate of fingertip loading during an isometric task

Risk factors for activity-related tendon disorders of the hand include applied force, duration, and rate of loading. Understanding the relationship between external loading conditions and internal tendon forces can elucidate their role in injury and rehabilitation. The goal of this investigation is to determine whether the rate of force applied at the fingertip affects in vivo forces in the flexor digitorum profundus (FDP) tendon and the flexor digitorum superficialis (FDS) tendon during an isometric task. Tendon forces, recorded with buckle force transducers, and fingertip forces were simultaneously measured during open carpal tunnel surgery as subjects (N=15) increased their fingertip force from 0 to 15N in 1, 3, and 10s. The rates of 1.5, 5, and 15N/s did not significantly affect FDP or FDS tendon to fingertip force ratios. For the same applied fingertip force, the FDP tendon generated more force than the FDS. The mean FDP to fingertip ratio was 2.4+/-0.7 while the FDS to tip ratio averaged 1.5+/-1.0 (p<0.01). The fine motor control needed to generate isometric force ramps at these specific loading rates probably required similar high activation levels of multiple finger muscles in order to stabilize the finger and control joint torques at the force rates studied. Therefore, for this task, no additional increase in muscle force was observed at higher rates. These findings suggest that for high precision, isometric pinch maneuvers under static finger conditions, tendon forces are independent of loading rate.     

Rearrangements of the T cell receptor delta gene in T acute lymphoblastic leukemia cells are distinct from those occurring in B lineage acute lymphoblastic leukemia and preferentially involve one V delta gene segment

Rearrangement of the TCR-delta gene was studied using J delta, C delta, and V delta probes in 61 cases of acute lymphoblastic leukemia (ALL) and several cases of chronic lymphoid neoplasms to define the specificity and the diversity of rearrangements occurring at the delta locus. TCR-delta rearrangements or deletions were found in all T (33 cases) and B lineage (28 cases) ALL but not in any case of B cell chronic proliferations (13 cases). The restriction patterns of rearrangement were clearly distinct between T and B ALL and use of one V delta probe showed that rearrangement of the V delta IDP2 gene segment which is also productively rearranged in the Peer cell line, occurred frequently in T-ALL but never in B lineage ALL. Studies of WT31 and delta TCS1 antibody reactivity showed that at least 4 of 13 CD3+ T-ALL cases expressed the delta protein. CD4 and/or CD8 Ag expression were observed in some of the gamma delta expressing T-ALL. These data show that particular TCR-delta gene rearrangements occur in neoplastic early B cells and that the combinatorial diversity of TCR-delta rearrangements in T cells is higher than initially expected. In addition this study shows that an important proportion of CD3 positive T-ALL cases express the gamma delta heterodimer.     

Application of pattern-mixture models to outcomes that are potentially missing not at random using pseudo maximum likelihood estimation

In this work, we fit pattern-mixture models to data sets with responses that are potentially missing not at random (MNAR, Little and Rubin, 1987). In estimating the regression parameters that are identifiable, we use the pseudo maximum likelihood method based on exponential families. This procedure provides consistent estimators when the mean structure is correctly specified for each pattern, with further information on the variance structure giving an efficient estimator. The proposed method can be used to handle a variety of continuous and discrete outcomes. A test built on this approach is also developed for model simplification in order to improve efficiency. Simulations are carried out to compare the proposed estimation procedure with other methods. In combination with sensitivity analysis, our approach can be used to fit parsimonious semi-parametric pattern-mixture models to outcomes that are potentially MNAR. We apply the proposed method to an epidemiologic cohort study to examine cognition decline among elderly.     

Alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development

Macrophages are abundant in the lower respiratory tract. They play a central role in the innate response to infection but may also modulate excessive inflammation. Both macrophages and ciliated epithelial cells respond to infection by releasing soluble mediators, leading to the recruitment of innate and adaptive effector cells. To study the role of lung macrophages in acute respiratory viral infection, we depleted them by the inhalation of clodronate liposomes in an established mouse model of respiratory syncytial virus (RSV) disease. Infection caused an immediate local release of inflammatory cytokines and chemokines, peaking on day 1, which was virtually abolished by clodronate liposome treatment. Macrophage depletion inhibited the activation (days 1 to 2) and recruitment (day 4) of natural killer (NK) cells and enhanced peak viral load in the lung (day 4). However, macrophage depletion did not affect the recruitment of activated CD4 or CD8 T cells, weight loss, or virus-induced changes in lung function. Therefore, lung macrophages play a central role in the early responses to viral infection but have remarkably little effect on the adaptive response occurring at the time of peak disease severity.     

Most of the yeast genomic sequences are not essential for cell growth and division

To determine the fraction of the yeast Saccharomyces cerevisiae genome that is required for normal cell growth and division, we constructed diploid strains that were heterozygous for random single disruptions. We monitored the effects of approximately 200 independent disruptions by sporulating the diploids and examining the phenotype of the resulting haploid strains. We found that only 12% of the disruptions were haploid-lethal, 14% resulted in slow growth, and an additional 4% were associated with some other new phenotype (such as an auxotrophic requirement). No obvious new phenotype was detected for 70% of the disruptions.