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Food Microbiology | Spotlight
Mediation Analysis as a Means of Identifying Dietary Components That Differentially Affect the Fecal Microbiota of Infants Weaned by Modified Baby-Led and Traditional Approaches
Claudia Leong, Jillian J. Haszard, Blair Lawley, Anna Otal, Rachael W. Taylor, Ewa A. Szymlek-Gay, Elizabeth A. Fleming, Lisa Daniels, Louise J. Fangupo, Gerald W. Tannock, Anne-Louise M. Heath
Charles M. Dozois, Editor
Claudia Leong
Department of Human Nutrition, University of Otago, Dunedin, New ZealandDepartment of Medicine, University of Otago, Dunedin, New Zealand
Jillian J. Haszard
Department of Human Nutrition, University of Otago, Dunedin, New Zealand
Blair Lawley
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
Anna Otal
Rachael W. Taylor
Department of Medicine, University of Otago, Dunedin, New ZealandMicrobiome Otago, University of Otago, Dunedin, New Zealand
Ewa A. Szymlek-Gay
Deakin University Institute for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Geelong, Australia
Elizabeth A. Fleming
Louise J. Fangupo
Gerald W. Tannock
Department of Microbiology and Immunology, University of Otago, Dunedin, New ZealandMicrobiome Otago, University of Otago, Dunedin, New Zealand
Anne-Louise M. Heath
Department of Human Nutrition, University of Otago, Dunedin, New ZealandMicrobiome Otago, University of Otago, Dunedin, New Zealand
Charles M. Dozois
INRS—Institut Armand-Frappier
The introduction of "solids" (i.e., complementary foods) to the milk-only diet in early infancy affects the development of the gut microbiota. The aim of this study was to determine whether a "baby-led" approach to complementary feeding that encourages the early introduction of an adult-type diet results in alterations of the gut microbiota composition compared to traditional spoon-feeding. The Baby-Led Introduction to SolidS (BLISS) study randomized 206 infants to BLISS (a modified version of baby-led weaning [BLW], the introduction of solids at 6 months of age, followed by self-feeding of family foods) or control (traditional spoon-feeding of purées) groups. Fecal microbiotas and 3-day weighed-diet records were analyzed for a subset of 74 infants at 7 and 12 months of age. The composition of the microbiota was determined by sequencing of 16S rRNA genes amplified by PCR from bulk DNA extracted from feces. Diet records were used to estimate food and dietary fiber intake. Alpha diversity (number of operational taxonomic units [OTUs]) was significantly lower in BLISS infants at 12 months of age (difference [95% confidence interval {CI}] of 31 OTUs [3.4 to 58.5]; P = 0.028), and while there were no significant differences between control and BLISS infants in relative abundances of Bifidobacteriaceae, Enterobacteriaceae, Veillonellaceae, Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, or Ruminococcaceae at 7 or 12 months of age, OTUs representing the genus Roseburia were less prevalent in BLISS microbiotas at 12 months. Mediation models demonstrated that the intake of "fruit and vegetables" and "dietary fiber" explained 29% and 25%, respectively, of the relationship between group (BLISS versus control) and alpha diversity.
IMPORTANCE The introduction of solid foods (complementary feeding or weaning) to infants leads to more-complex compositions of microbial communities (microbiota or microbiome) in the gut. In baby-led weaning (BLW), infants are given only finger foods that they can pick up and feed themselves—there is no parental spoon-feeding of puréed baby foods—and infants are encouraged to eat family meals. BLW is a new approach to infant feeding that is increasing in popularity in the United States, New Zealand, the United Kingdom, and Canada. We used mediation modeling, commonly used in health research but not in microbiota studies until now, to identify particular dietary components that affected the development of the infant gut microbiota.
The first year of life sees marked changes in the composition of the gut microbiota of infants as the diet changes from one that is entirely milk based to one that is similar to the family diet by 12 months or so of age (1). Although attention has been paid to the impact of breast milk and infant formula on the composition of the microbiota (2–8), less research has investigated the impact of the change in diet that occurs when infants are introduced to solid foods during the complementary feeding period (known in some countries as "weaning") (9). To date, the majority of studies on this transition have been observational (10, 11), and the few randomized controlled trials (RCTs) have focused largely on the impact of iron fortification on the gut microbiota (12, 13).
Traditionally, parents have been encouraged to start spoon-feeding their infant puréed foods from around 6 months of age, progressing to mashed and then chopped foods in the hope that they will be eating family foods by around 12 months of age (1). However, an alternative method of complementary feeding, known as baby-led weaning (BLW), is becoming popular in New Zealand (14), the United Kingdom (15), the United States (16), and Canada (17). In BLW, infants are encouraged to feed themselves whole pieces of food from the family meal from 6 months of age, instead of being offered "baby food" (18). As a result, infants following BLW are more likely to eat the same foods as the rest of the family than their traditionally fed counterparts (14). Because the introduction of family foods is a major determinant of the development of the gut microbiota (19), it is possible that the more rapid transition to the family diet observed in baby-led approaches will differentially influence the gut microbiota in these children.
The Baby-Led Introduction to SolidS (BLISS) randomized controlled trial (20, 21) has investigated the impact of a modified version of BLW on growth (22), choking risk (23), and iron status (24) and provides an opportunity to investigate the impact of the early introduction of an adult-type diet on the developing infant gut microbiota. The aim of this study was to determine whether a "baby-led" approach to complementary feeding affects the fecal microbiota composition relative to traditional spoon-feeding and, if so, to use mediation analysis to associate dietary components with altered microbiota.
Study population.Fecal samples were obtained from 73 participants at 7 months of age (data for 1 BLISS participant are missing) and 68 participants at 12 months of age (data for 3 control and 3 BLISS participants are missing) (Fig. 1). The baseline and early feeding characteristics of these participants are shown in Tables 1 and 2; infants were predominantly born by vaginal delivery to university-educated mothers. BLISS infants were introduced to complementary foods later than control infants. No differences were observed in the rates of breastfeeding, or the amounts of breast milk or infant formula consumed, at 7 or 12 months of age. Rates of recent antibiotic use were similar in control and BLISS infants at 7 months (16.7% versus 13.5%; P = 0.707) and 12 months (16.1% versus 8.8%; P = 0.371) of age. From the main-outcomes paper, BLISS infants had very good adherence to the baby-led approach and were significantly (P < 0.01) more likely to feed themselves most or all their food than control infants at every age (for example, 74% of BLISS infants at 7 months of age compared to 21% of control infants) (22). Using population-averaged generalized estimating equations with the full sample, BLISS infants were also significantly more likely to be eating the same foods as the rest of their family by 7 months of age, having 3.3 times the odds (95% confidence interval [CI], 2.0, 5.6; P < 0.001) of eating the same evening meal as the rest of the family compared to the control infants (data not shown).
Flow diagram for the microbiota component of the BLISS (Baby-Led Introduction to SolidS) study.
Characteristics of the BLISS study participants who agreed to provide a fecal samplea
Early diet characteristics of the BLISS study participants who agreed to provide a fecal samplea
Gut microbiota.Figure 2 shows that alpha diversity (all samples rarefied to 30,000 sequences per sample), as measured by observed "species," increased in both groups from 7 to 12 months of age (mean change [95% CI] of 77 OTUs [64, 90]; P < 0.001 from a paired t test), and at 12 months, the BLISS group had significantly lower alpha diversity than the control infants (P = 0.028). As shown in Table 3, no significant group differences were observed for the five measures of alpha diversity at 7 months (all P > 0.1), and the proportion of variance explained by group, parity, and maternal university education was low for observed species, phylogenetic diversity, and the Chao1 estimator (all <2%). For those measures of alpha diversity that included evenness (Simpson index and Shannon index), the proportion of variance explained was slightly higher (6.3% and 8.1%, respectively) although still low.
Rarefaction curves of observed OTUs against the number of sequences from control and BLISS groups at 7 and 12 months of age. OTUs, operational taxonomic units (a proxy for observed species and a measure of alpha diversity). *, P value between groups at 12 months.
Alpha diversity measures for control and BLISS groups at 7 and 12 months
At 12 months of age, however, the BLISS group had significantly lower alpha diversity than the control group. The BLISS group had a mean of 31 fewer operational taxonomic units (OTUs) (P = 0.028) and a 0.52-lower Shannon index (P = 0.006) than the control group. The proportion of variance explained at 12 months (between 16% and 21%) was higher than that at 7 months (between 0.4% and 8%) (Table 3). As observed species and Shannon index were the alpha diversity measures that covered richness as well as richness and evenness with the highest R2 values and had the strongest associations with the randomized group, subsequent analyses with alpha diversity were limited to these two measures.
Sensitivity analyses were carried out with the inclusion of antibiotic use in the days before the fecal sample was collected (n = 11 at 7 months; n = 8 at 12 months). No appreciable impact on the results at 7 months was seen, but the results at 12 months were slightly strengthened by the inclusion of antibiotic use in the model (mean difference [95% CI] for observed species of −33 OTUs [−62, −5] [R2 = 0.19]; mean difference [95% CI] for Shannon index values of −0.56 [−0.94, −0.19] [R2 = 0.23]).
Figure 3 illustrates the relative abundances of the 7 most abundant families (median of at least a 1% relative abundance), which in combination explained approximately 86% of the sequence data. There were no significant differences in the relative abundances of the 7 bacterial families between groups at either 7 or 12 months of age (see Table S1 in the supplemental material). However, for all infants combined, there was a decrease in the relative abundances of Bifidobacteriaceae, Enterobacteriaceae, and Veillonellaceae, with increases in the relative abundances of Lachnospiraceae and Ruminococcaceae at between 7 and 12 months of age (Table S2).
Relative abundances of the top (>1%) seven bacterial families in feces of control and BLISS infants at 7 and 12 months of age.
Beta diversity metrics (Bray-Curtis dissimilarity unweighted UniFrac, weighted UniFrac, and Jaccard distance) were determined, and permutational multivariate analysis of variance (PERMANOVA) was used to compare diet/time groups for each metric. The results indicated that the BLISS and control group microbiotas were different at 12 months but only when using presence/absence data (Jaccard distance and unweighted UniFrac; P = 0.028 and 0.037 and q = 0.0336 and 0.0444, respectively).
While it is tempting to think that if alpha and beta diversities differ between groups, there will be corresponding clear-cut differences in OTU distributions, this may not be the case because multiple communities may differ in diversity without having taxonomic differences in common. Nevertheless, we attempted to identify OTUs that were differentially represented in the control group relative to the BLISS group at 7 and 12 months of age using analysis of composition of microbiomes (ANCOM), gneiss, and multivariate association with linear models (MaAsLin). The original OTU table was split into two tables separated by sampling time in order to focus on differences due to diet rather than developmental age. Additionally, the full OTU table was retained where models could be applied to account for continuous variables (in this case, "time") When the tables were split, no OTUs were reported to be differentially abundant for either time regardless of the method. When MaAsLin was employed with the full table and time was included as a forced predictor, six OTUs were predicted to be differentially abundant between the dietary groups (Table 4), and all were less abundant in the BLISS group. Three of these OTUs represented the genus Roseburia.
OTUs differentially abundant according to diet when controlling for timea
An alternative approach was to search for differential prevalence by analyzing the OTU table using presence/absence data. A between-group difference of 10 participants with the presence of a species was decided to be a meaningful difference for this analytical approach (i.e., 10 more participants in the control group had a particular OTU present than in the BLISS group; this difference also represents the difference that we would give 80% power for detection to the 5% level). When applied to an OTU-level table, at 12 months, 21 OTUs were overrepresented in the control group, while two OTUs were overrepresented in the BLISS group. All OTUs in the control group were assigned to the order Clostridiales, and 7 OTUs were classified as belonging to the genus Roseburia. At 7 months, only three OTUs were overrepresented in the control group (Table S3).
Diet.Energy intakes were similar in control and BLISS participants at 7 months of age (means [standard deviations {SD}] of 2,820 (470) kJ/day and 2,860 (440) kJ/day, respectively; P = 0.749) and also at 12 months of age (means [SD] of 3,460 (850) kJ/day and 3,430 [480] kJ/day, respectively; P = 0.881). However, the proportion of energy contributed by each food group changed considerably over time (Fig. 4). Milk (breast milk and infant formula) contributed approximately 78% of the energy for both groups at 7 months of age, decreasing to around 36% at 12 months.
Proportions of total energy intake contributed by nine food groups at 7 and 12 months of age.
Predictors of alpha diversity.Table 5 shows that the only variable associated with alpha diversity at 12 months of age, from a range of demographic variables, feeding characteristics, and antibiotic use, was parity: greater alpha diversity was observed in children with a higher birth order. The intake of "breads and cereals," "fruit and vegetables," and "dietary fiber" at 7 months of age were all positively and significantly associated with at least one measure of (higher) alpha diversity at 12 months of age (Table 6). For example, at 7 months, the intake of an additional 10 g of breads and cereals was associated with 9.5 more OTUs and a 0.14-higher Shannon index at 12 months. "Infant milk" and "meat" intake were also moderately associated with alpha diversity but not statistically significantly (P = 0.066 and 0.0.089, respectively, for the number of OTUs and P = 0.234 and 0.070, respectively, for the Shannon index).
Associations between alpha diversity (number of OTUs and Shannon index) and variables related to demography, change in feeding mode, and antibiotic use at 12 months (n = 68)
Associations between dietary components at 7 months and alpha diversity at 12 months (number of OTUs and Shannon index) (n = 63)
Because there were significant associations between diet at 7 months of age and alpha diversity at 12 months, we hypothesized that diet at 7 months might explain (mediate) the observed differences in alpha diversity between the BLISS and control groups. However, as there were no differences between groups for infant milk intake (mean difference [95% CI] of −4 [−73 to 65] g/day), bread and cereal intake (mean difference [95% CI] of 1.9 [−5.9 to 9.8] g/day), or meat intake (mean difference [95% CI] of −0.9 [−6.0 to 4.1] g/day), these foods were not considered to be mediators, leaving only the intake of fruit and vegetables and dietary fiber as potential mediators. Figure 5 illustrates the mediating effect that the intake of fruit and vegetables and dietary fiber at 7 months had on the relationship between group and alpha diversity at 12 months. The control group consumed a mean of 53 g/day more fruit and vegetables than the BLISS group, and this difference explained 29% and 27% of the associations between group and observed species and the Shannon index, respectively. Similarly, the control group consumed a mean of 1.3 g/day more fiber than the BLISS group, explaining 25% of the relationship between group and both observed species and the Shannon index. We also investigated the association of dietary intake at 12 months with alpha diversity; only the intake of breast milk and sweet foods showed a meaningful association, but these variables were not different between the groups and thus were unlikely to explain a meaningful proportion of the differences seen between the groups at 12 months.
Observed relationship between infant group and alpha diversity (mediation model). (A) Fruit and vegetables (10 g for each). (B) Dietary fiber (for each gram) intake. (n = 63) (see Fig. S1A in the supplemental material for the key).
Microbial composition analysis using several alpha diversity measures revealed that infants following BLW had significantly lower alpha diversity at 12 months of age than control infants. Using mediation analysis, a novel approach in microbiota studies, to explore the association between group and alpha diversity, we were able to show that 29% and 25% of the links between group and alpha diversity at 12 months could be explained by lower intake of fruit and vegetables and dietary fiber in the BLISS group at 7 months.
Alpha diversity increased from 7 months to 12 months of age in both groups, as expected due to the introduction of sources of food for the gut microbiota other than milk (13, 25). We also noted decreases in the relative abundances of Bifidobacteriaceae, Enterobacteriaceae, and Veillonellaceae and increases in the relative abundances of Lachnospiraceae and Ruminococcaceae from 7 to 12 months. This is in agreement with data from previous studies (3, 26, 27). The substantial decrease in infant milk intake from 7 to 12 months likely explains the relative decreases in the abundances of Bifidobacteriaceae, which utilize lactose and human milk oligosaccharides that are found in milks (28), and Veillonellaceae, which have species that are able to utilize lactate, a fermentation product of Bifidobacteriaceae (29). The relative increases in the abundances of Lachnospiraceae and Ruminococcaceae at 12 months can be attributed to the introduction of solid foods, as both Lachnospiraceae and Ruminococcaceae have species that are known to play a role in polysaccharide (i.e., "complex carbohydrate") degradation (30). They form the most abundant bacterial families of the adult fecal microbiota in Western populations.
At 12 months of age, infants in the BLISS group had lower bacterial alpha diversity than those in the control group. This was explained, at least in part, by the differential prevalence of OTUs representing members of the genus Roseburia, a taxon whose abundance in the bowel is known to be associated with the amount of dietary fiber consumed by the human host (31, 32). This observation therefore supported the association between alpha diversity and dietary fiber that was revealed by mediation analysis. The BLISS infants were encouraged to eat the same foods as the rest of the family (i.e., a more adult diet) from the start of complementary feeding, and we expected that this would result in greater alpha diversity because a recent study showed that the progression to family foods was strongly associated with increased alpha diversity (observed species and Shannon index) (19). Our results were sustained even after adjustment for parity, which has been found to be significantly associated with alpha diversity in the present study and elsewhere (33), presumably as a result of exposure to a wider range of bacteria from the range of education and other environments with which siblings interact, nor can these findings be explained by mode of delivery (34), breast milk intake, or recent use of antibiotics (35) which were shown previously to be associated with lower alpha diversity but were not significant predictors of alpha diversity in our study.
To reveal the mechanism by which the BLISS intervention was associated with lower alpha diversity, we conducted mediation analyses. Mediation analysis is increasingly being encouraged in the health literature to elucidate the underlying pathways of effects on health outcomes (36, 37). Mediation analysis enhances causal inference and allows the identification of specific components of an intervention that were effective. Mediation analysis has been used in previous randomized controlled trials, where an intervention that involved changing behaviors (such as a way of eating) was associated with an outcome (such as a difference in food intake) (38, 39). To then understand why the intervention affected the outcome, mediation analysis was used to identify the mediators (such as the use of certain maternal feeding practices or a child's access to noncore food) (36–39). Mediation analysis is therefore well suited to evaluate the specific dietary mechanisms that might explain a difference in the gut microbiota as the result of a complex behavioral intervention, such as in the BLISS study. For mediation to be present, the independent variable (in this case, randomized group) must affect both the outcome (in this case, alpha diversity) and the mediating variable (e.g., food intake), the mediating variable must affect the outcome, and the relationship between the independent variable and the outcome must be reduced in magnitude when the mediating variable is controlled for (40, 41). Temporal ordering should be present. In our study, we found that intakes of fruit and vegetables and dietary fiber were mediators explaining 29% and 25%, respectively, of the links between group and alpha diversity. Even though the intake of breads and cereals was associated with alpha diversity, the control and BLISS groups did not have significantly different intakes of this group, meaning that it was not a mediator of the association between group and alpha diversity. These findings suggest that at least one-quarter of the impact of the BLISS intervention on alpha diversity can be explained by the lower intake of fruit, vegetables, and dietary fiber in the BLISS group at 7 months of age. Dietary fiber has been shown to be positively associated with alpha diversity measures in this age group (19), presumably because the range of polysaccharides that are indigestible to the host (i.e., dietary fiber) provide growth substrates for a range of gut microbes.
The significantly lower intake of fruit and vegetables and of dietary fiber among the BLISS infants is an interesting finding. Although there has been an expectation that following a baby-led approach to infant feeding would result in the consumption of a wider variety of healthy foods (18), concerns have been expressed that the handheld foods eaten by adults are not necessarily "healthier" (42). The infants in the BLISS group were introduced to solids approximately 3 weeks later than the control infants, so at 7 months of age, they were earlier in their complementary feeding journey than controls; perhaps this resulted in them being offered less fruit and vegetables. Moreover, one small pilot study reported that parents who were following BLW with their infant had higher-than-recommended intakes of saturated fat, sodium, and sugar (43), suggesting that if infants were being given the foods that their parents were eating, they may be offered a diet that is less healthy and therefore probably lower in fruit and vegetables. However, a more likely explanation for the lower intake of fruit, vegetables, and dietary fiber is that infants who are being traditionally weaned are usually spoon-fed purées in the early months of complementary feeding, and these are commonly based on fruit and vegetables because they can be blended to a smooth consistency and are often sweet and therefore highly palatable. In fact, in the United Kingdom, about two-thirds of all baby foods have been reported to contain fruit, vegetables, or both (44).
Major strengths of our study were the use of a randomized controlled design that ensured that differences between the groups were due to the intervention rather than differences in participant characteristics and the use of 3-day weighed-diet records to provide detailed high-quality dietary data.
This study shows that infants following BLW consume a more adult-type diet and have a fecal microbiota with a less complex composition at 12 months than infants following traditional spoon-feeding. Lower intakes of fruit and vegetables and of dietary fiber are partially responsible for this lower alpha diversity. The difference in alpha diversity between groups is modest and, at this stage, cannot be related to changes in child development or health. Larger, longer-term studies are required before any conclusions can be made about the possible impact of these differences or whether infant feeding guidelines should recommend that infants following BLW consume more fruit and vegetables or dietary fiber than is currently the case.
Study design.The BLISS study was a 2-year randomized controlled human intervention trial (Australian New Zealand Clinical Trials Registry number ACTRN12612001133820) that compared a version of BLW (the infants feed themselves family foods) modified to address concerns about choking, iron deficiency, and growth faltering (42) with a control group (receiving usual care, predominantly traditional spoon-feeding). Because the protocol (21), pilot study (20), and main-outcomes paper (22) have been reported, only relevant information is included here. The Lower South Regional Ethics Committee of New Zealand and the University of Otago Human Ethics Committee approved the study, and written informed consent was obtained from all adult participants before randomization and before collection of the fecal samples for gut microbiota analysis. Fecal samples collected at 7 and 12 months of age and a 3-day weighed-diet record collected at 7 months were used to determine the impact of the BLISS intervention on changes in alpha diversity and the relative abundances of bacterial families in the infant gut microbiota.
Participants.All women in late pregnancy who were booked into the only maternity hospital in Dunedin, New Zealand, from December 2012 to March 2014 were eligible to participate in the BLISS study if they were at least 16 years old and booked before 34 weeks of gestation. Infants were excluded if they were born preterm or had a congenital abnormality likely to affect feeding or growth. Participants were randomized to the control (n = 106) or BLISS (n = 108) group via random-length blocks (maximum of 7) after stratification for parity (first child or subsequent child) and maternal education (nontertiary or tertiary). Allocation was concealed by using opaque presealed envelopes. After birth, 8 infants were excluded, so the final samples included 101 control and 105 BLISS participants (Fig. 1).
Fecal samples for microbiota analysis were collected from a subset of the infants (Fig. 1). Of the 165 participants in the BLISS study at the time of fecal collection, 86 were 7 months of age or younger, 74 (86%) of whom had parents who agreed to provide a fecal sample for microbiota analysis. There were no significant differences in maternal age, body mass index (BMI), parity, or education; household deprivation; or the sex or birth weight of the child between participants who did (n = 74) and those who did not (n = 132) provide a fecal sample (statistical tests for groups as stated in "Statistical analysis," below). Therefore, the subset was representative of the BLISS study cohort as a whole.
Intervention.Both the control group and the BLISS group had access to government-funded routine midwifery and well-child care (45, 46). The BLISS group had eight additional contacts from pregnancy to 9 months of age. These contacts provided education and support to prolong milk feeding (ideally exclusive breastfeeding) and delay the introduction of complementary foods until 6 months of age (provided by an international board-certified lactation consultant) and specific advice at 5.5, 7, and 9 months on how to follow the BLISS approach to infant feeding (provided by trained researchers) (20, 21). The key components of the BLISS approach were to provide foods in a form that the babies can pick up themselves; to let babies feed themselves; to offer an iron-rich food, an energy-rich food, and an easy-to-eat food with every meal; and to eat as a family whenever possible (20, 21). Adherence to a baby-led approach was defined as the infants feeding themselves most or all their food in the previous week (21) and was measured by questionnaire at 6, 7, 8, 9, and 12 months of age.
Demographic data.Information on infant sex, birth weight, parity, and level of household deprivation (47) was obtained from hospital records. Self-reported prepregnancy height and weight, maternal education attainment, and maternal age were obtained from baseline questionnaires that were completed before randomization in late pregnancy. The age when complementary foods were first introduced was determined prospectively by using data from brief feeding questionnaires administered at 2, 4, 6, 7, 8, 9, and 12 months of age (21).
Gut microbiota data.Parents were asked to collect a fecal sample from their infant's diaper at 7 and 12 months of age. The sample was then stored in the home freezer (−18°C) in study-provided freezer containers before collection and delivery to the University of Otago Department of Microbiology and Immunology, where they were stored at −80°C until DNA extraction. DNA was extracted from 250 mg feces according to the kit protocol provided by the manufacturer (PowerSoil DNA isolation kit, catalog number 12855-100; Mo Bio). Amplification of the 16S rRNA gene V4 region, library preparation, and sequencing were carried out at Argonne National Laboratories (University of Chicago) using 2- by 250-bp paired-end reads on an Illumina MiSeq instrument. Sequences were analyzed by using a combination of QIIME version 1.9.1 (48) and vsearch version 1.9.5 (49). Taxonomy classifications were made by using the SILVA version 123 database (50).
Microbiota composition was described by using five alpha diversity measures: the number of operational taxonomic units (OTUs) (a proxy for observed species), phylogenetic diversity, the Chao1 estimator, the Simpson index, and the Shannon index. Therefore, three indices described microbial richness alone (i.e., number of species), observed species, phylogenetic diversity, and the Chao1 estimator, and two indices described richness and evenness (i.e., the equality of distribution of the species' frequencies), the Simpson index and Shannon index. The OTU table was rarefied to the minimum sample count (30,000 sequences) for calculation of the alpha diversity measures. Relative abundance at the family level was calculated by collapsing the raw OTU table based on seven-level taxonomy strings obtained from the SILVA version 123 database.
Beta diversity metrics (Bray-Curtis index, unweighted UniFrac, weighted UniFrac, and Jaccard distance) were applied by using the QIIME2 (https://qiime2.org/) command line interface (v.2018.4) and the core-metrics-phylogenetic plug-in with a sampling depth of 30,000 sequences. Group significance for each metric was measured with PERMANOVA (51).
Differential abundance testing was carried out on the full OTU table and on OTU tables that were filtered to contain data from either the 7-month samples or the 12-month samples in order to concentrate on differences between the dietary groups at distinct time points. ANCOM (52) and GNEISS (53) tests were run through the QIIME2 command line interface using default parameters. More specifically, the diet metadata group was analyzed for ANCOM, and for GNEISS analysis, "Diet+Time" was used for modeling ordinary least-squares regression (OLS-regression) on the full OTU table.
Differential abundance was also tested by using MaAsLin v.0.0.5 (54) using OTU tables filtered to 7-month and 12-month time points and on the full OTU table with "time" included as a forced predictor.
Dietary data.Parents completed a 3-day weighed-diet record on 3 randomly assigned nonconsecutive days (1 weekend day and 2 weekdays) over 3 weeks. Parents were taught how to complete the weighed-diet record by an interviewer, given an opportunity to practice weighing a food with the interviewer's support, given detailed written instructions and examples to take home with them, and instructed to contact the researchers if they had any questions while completing the record. Breast milk intake was estimated by using total daily volumes of 750 g/day at 7 months of age and 448 g/day at 12 months (55). For infants who were fed both breast milk and infant formula, the amount of infant formula consumed was subtracted from 750 g/day at 7 months and 448 g/day at 12 months, with the remainder being entered as breast milk. Diet records were analyzed by using the Kai-culator program (version 1.13s; University of Otago), which uses data from the New Zealand Food Composition Database (FOODfiles 2010), nutrient data for commonly consumed recipes from national nutrition survey data, and nutrient data for commercial infant foods collected by the research team (21).
Nine food groups were defined based on food groups from the New Zealand Ministry of Health Food and Nutrition Guidelines for Healthy Infants and Toddlers (1) and the number of consumers (i.e., at least eight consumers were required in each food group so that there was sufficient power to perform the food group analyses): "breast milk," "infant formula," "dairy," "fruit and vegetables," "breads and cereals," "meat, fish, and poultry," "sweet food," "savory food," and "miscellaneous." Ingredients in recipes were coded into their specific food groups instead of the whole recipe being assigned to a diverse "mixed dishes" category.
Statistical analysis.Data were analyzed by using Stata software (version 13; StataCorp). To compare antibiotic use, feeding behaviors, and infant formula consumption at 7 months of age between randomized groups, a chi-squared test was used. A log rank test (i.e., time to events) was used to compare the ages of introduction of formula, solids, and infant cereal (Table 2). To determine differences in alpha diversity between groups at both 7 and 12 months, linear regression was used, with adjustment for parity and maternal education (i.e., the stratification variables). Mean differences, 95% confidence intervals (CI), and P values were calculated along with the proportion of variance explained (R2) by the independent variables (Fig. 2 and Table 3). Subsequent analyses for alpha diversity were then limited to one measure that described richness only and one measure that described richness and evenness. The two measures (number of OTUs and Shannon index) were chosen because they had the highest R2 values (i.e., they explained the highest proportion of the variance) and had the strongest association with randomized group (Table 3).
Group and age differences with respect to the relative abundances of bacterial families for which the median relative abundance was >1% were determined by using median regression (Fig. 3; see also Tables S1 and S2 in the supplemental material).
Linear regression was also used to assess whether demographic and feeding variables and antibiotic use in the week before fecal sampling were related to alpha diversity, with adjustment for randomized group (Table 6). Thereafter, the factor that was found to be significantly associated with alpha diversity was considered a confounder and adjusted for in subsequent analyses. To explore whether dietary components at 7 months of age predicted alpha diversity at 12 months, regression models were generated and adjusted for randomized group, parity (confounder from Table 5), and maternal education (Table 5).
Mediation analysis was then used to determine the extent to which the association between randomized group and alpha diversity was due to differences in food group intake; i.e., food group intake "mediated" the association (Fig. S1). The requirements for mediation were considered to exist (36) if randomized group predicted alpha diversity at 12 months of age, and food group intake at 7 months was both related to alpha diversity at 12 months and different between the randomized groups. The "proportion mediated" was the proportion of the effect size without the mediator ("total effect") that was reduced when the mediator was included in the regression ("direct effect"). Decisions and explanations for mediation analysis were based on both effect sizes and P values. Residuals for all linear regression models were plotted and visually assessed for homogeneity of variance and normality.
Accession number(s).The DNA sequence data have been deposited in the NCBI database under BioProject accession number PRJNA419227.
We acknowledge the members of the BLISS study team who contributed to data collection. We also thank the BLISS study participants.
We thank Lottery Health Research, Meat & Livestock Australia, the Karitane Products Society, Perpetual Trustees, The New Zealand Women's Institute, and the University of Otago, with "in kind" contributions from Heinz Watties Ltd. C.L. is supported by a University of Otago doctoral scholarship, and R.W.T. is supported by a fellowship from the Karitane Products Society that is paid via the University of Otago. The funding sources had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication.
We declare that there is no conflict of interest.
Received 17 April 2018.
Accepted 2 July 2018.
Accepted manuscript posted online 13 July 2018.
Address correspondence to Gerald W. Tannock, gerald.tannock{at}otago.ac.nz.
↵* Present address: Anna Otal, Stemcell Technologies, Vancouver, BC, Canada.
Citation Leong C, Haszard JJ, Lawley B, Otal A, Taylor RW, Szymlek-Gay EA, Fleming EA, Daniels L, Fangupo LJ, Tannock GW, Heath A-LM. 2018. Mediation analysis as a means of identifying dietary components that differentially affect the fecal microbiota of infants weaned by modified baby-led and traditional approaches. Appl Environ Microbiol 84:e00914-18. https://doi.org/10.1128/AEM.00914-18.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.00914-18.
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Applied and Environmental Microbiology Aug 2018, 84 (18) e00914-18; DOI: 10.1128/AEM.00914-18
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All Greenwich London Borough Schools to Close for Xmas Holidays
By Tony Winterburn • 14 December 2020 • 6:53
All Greenwich London Borough Schools to Close for Xmas Holidays.
Greenwich council has ordered all schools in the area to close for the Christmas holidays after a surge in Covid cases was reported. The council has been told the spike in coronavirus cases "demands immediate attention" after new infections went above 200 per 100,000 people.
All 133 schools across the borough will now close on Monday, Dec. 14, instead of on Thursday, which was meant to be the last day before the term ends for the Christmas holidays. The news comes as London could likely move into Tier 3 restrictions when the situation is reviewed on Wednesday, Dec. 16.
Public Health England advised Greenwich council to shut schools early because the coronavirus situation was escalating "extremely quickly", Council leader, Danny Thorpe, said, quote: "The situation in Greenwich in relation to Covid-19 is now escalating extremely quickly. I have today been briefed by colleagues from Public Health England that the pandemic in Greenwich is now showing signs that we are in a period of exponential growth that demands immediate action.
"We now have the highest rates of infection in Greenwich than at any time since March. For these reasons, I have therefore asked all schools in Greenwich to close their premises from Monday evening and move to online learning for the duration of the term, with the exception of key worker children and those with specific needs (exactly the same as in the first lockdown)."
Thank you for taking the time to read this news article "All Greenwich London Borough Schools to Close for Xmas Holidays". For more UK daily news, Spanish daily news and Global news stories, visit the Euro Weekly News home page.
Tony Winterburn

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Our recent Education Review Office Report is now available online and we are pleased to share this with our school community. The report affirms our focus on achievement outcomes and recognises the ongoing success of our students.
ERO's confidence in the school is demonstrated by the awarding of a four to five year review cycle, the highest possible level.


ELECTED OFFICIALS NEED TO MEASURE THEIR WORDS by Bob Shannon | Virginia Right!
As the school budget debate heats up and the newly elected members of the KW B.O.S weigh in on the topic, voting this past week on a approach that allows the B.O.S to channel funds into specific categories ( the measure lost 3-2) School Board Member Kathy Morrison made the public statement "we've done nothing to deserve this", referencing of course the measures intended effect , to instill tighter controls over how funds are spent within KW Schools. Of course the defenders of the status quo will support Ms. Morrison's statement, and setting aside the self -serving interests of those who benefit by current practice of unfettered spending, let's take a look at it from another point of view—-the taxpayers.
All of 2015 the KWTP made it a major point to contrast the KW Schools spending some $90,000 a week more than Nottoway County Schools, with some 300 more students and 2 more schools than KW's 4. This is a very generous comparison since our analysis excluded the 2.3 million KW Schools spent last year on just debt service ( Nottoway allocated $230,000.00 for debt service, 1/10 th the amount spent here).
In 2014 our group distributed copies ( Ms. Morrison and all SB Members received a copy ) of the Heritage Foundations report entitled " The School Staffing Surge" which documents over a period from 1950—2009 the increases in the number of students ( 96% ) Teachers ( 252%) and Administrative Staffing ( 702%) This data has also been prominently featured on a number of our beloved yellow signs over the last year.
To date not a single member of the school board, nor the former Superintendent has said a word about the Heritage Foundations report. Many elected officials , to include members of the previous B.O.S admit to not even reading it . One might ask an obvious question as to why the side always advocating for more money and higher school budgets always gets an audience, when a contrarian point of view doesn't even merit examination ? It is a fair question. Since the issue of the additional $90,000.00 a week that KW Schools spend above and beyond a system larger than our own was also so prominently featured all of 2015 why not any objections or push back from Ms. Morrison on that point ?
"we've done nothing to deserve this" If Novembers election results mean anything, taxpayers think otherwise Ms. Morrison.
the folks can make line item comparisons between the two systems. Very Interesting!
Thank you for making this very relevant point, something I was not aware of. Can you elaborate a bit, when was this passed and how will it work. Where would one find these comparisons ? Has Virginia Government a website this will be posted on. Tell us more please.


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Fabinho: Liverpool midfielder ruled out of FA Cup final with hamstring injury
No Comments on Fabinho: Liverpool midfielder ruled out of FA Cup final with hamstring injury
Fabinho has played 47 games for Liverpool this season, including three FA Cup ties in which he scored two goals
Venue: Wembley Stadium Date: Saturday, 14 May Kick-off: 16:45 BST
Coverage: Watch on BBC One, BBC iPlayer and BBC Sport website
Liverpool manager Jurgen Klopp has confirmed his side will be without midfielder Fabinho for Saturday's FA Cup final against Chelsea.
The Brazilian came off with a hamstring injury in Tuesday's win at Aston Villa.
Fabinho will also miss the final two league games of the season, but Klopp hopes he will be fit in time for the Champions League final on 28 May.
"We can cope," Klopp said, when asked how the 28-year-old's absence would impact Liverpool's title hopes.
"We have to. Having him or not having him is a difference but it is all fine. That's normal stuff that happens. It's never in a position where you have 12 options, it's always in a position where you don't."
Klopp added: "There's a good chance that he will be available for the Champions League final."
Jordan Henderson came off the bench to replace Fabinho in the comeback victory at Villa Park and Klopp hinted that Henderson will retain the defensive midfield position at Wembley.
"Hendo came on and played incredible. He has played super games this season at number six, so that is not a problem."
A vital cog in the Liverpool machine, the Reds will be without Fabinho for the Premier League games against Southampton and Wolves as they hope for a Manchester City slip-up.
Liverpool are second, three points behind defending champions City and with a significantly lower goal difference with two games remaining.
Venue:Wembley StadiumDate:Saturday, 14 MayKick-off:16:45 BST
Coverage:Watch on BBC One, BBC iPlayer and BBC Sport website
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These awards are for students whose endeavors and accomplishments reflect the objectives of Alpha Pi Mu, namely scholarship and service to the Department, the College, and the University.
This award is for a senior whose activities and accomplishments demonstrate excellence in leadership.
The applications for these awards are available using the links above. Please complete the applications by Wednesday, March 6, at 4:00 p.m. If you have any questions, please feel free to contact Dr. Kimberly Ellis at kpellis@vt.edu.
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Cosmegen is indicated for the treatment of adult and pediatric patients with Wilms tumor, as part of a multi-phase, combination chemotherapy regimen.
Cosmegen is indicated for the treatment of adult and pediatric patients with rhabdomyosarcoma, as part of a multi-phase, combination chemotherapy regimen.
Cosmegen is indicated for the treatment of adult and pediatric patients with Ewing sarcoma, as part of a multi-phase, combination chemotherapy regimen.
Cosmegen is indicated for the treatment of adult and pediatric patients with metastatic, nonseminomatous testicular cancer, as part of a multi-phase, combination chemotherapy regimen.
Cosmegen is indicated for the treatment of post-menarchal patients with gestational trophoblastic neoplasia, as a single agent or as part of a combination chemotherapy regimen.
Cosmegen is indicated for the treatment of adult patients with locally recurrent or locoregional solid malignancies, as a component of palliative or adjunctive regional perfusion.
The recommended dose of Cosmegen, as part of a multi-agent combination chemotherapy regimen, is 45 mcg/kg intravenously once every 3 to 6 weeks for up to 26 weeks.
The recommended dose of Cosmegen, as part of a multi-agent combination chemotherapy regimen, is 15 mcg/kg intravenously once daily for 5 days every 3 to 9 weeks for up to 112 weeks.
The recommended dose of Cosmegen, as part of a multi-agent combination chemotherapy regimen, is 1250 mcg/m 2 intravenously once every 3 weeks for 51 weeks.
The recommended dose of COSEMGEN, as part of a cisplatin-based, multi-agent combination chemotherapy regimen, is 1000 mcg/m 2 intravenously once every 3 weeks for 12 weeks.
The recommended dose of COSEMEGEN for nonmetastatic and low-risk metastatic disease is 12 mcg/kg intravenously daily for five days as a single agent.
The recommended dose of COSEMEGEN, as part of a multi-agent combination chemotherapy regimen, for high-risk metastatic disease is 500 mcg intravenously on Days 1 and 2 every 2 weeks for up to 8 weeks.
The recommended dose of Cosmegen, in combination with melphalan, is 50 mcg/kg once for lower extremity or pelvis.
The recommended dose of Cosmegen, in combination with melphalan, is 35 mcg/kg once for upper extremity.
Calculate the dose for obese or edematous patients based on ideal body weight.
Visually inspect the vials for particulate matter and discoloration, whenever solution and container permit.
Reconstitute each vial by adding 1.1 mL of Sterile Water for Injection without preservative using aseptic techniques.
The reconstituted product should be a clear, gold-colored solution at a concentration of 500 mcg/mL.
Further dilute the reconstituted product with 5% Dextrose Injection or 0.9% Sodium Chloride Injection to yield concentrations greater than 10 mcg/mL.
Store at room temperature for no more than 4 hours from reconstitution to completion of injection or infusion. Discard after 4 hours.
Cosmegen does not contain a preservative. Discard any unused portions.
Administer the diluted reconstituted product intravenously over 10 to 15 minutes.
Do not use in-line filters with a cellulose ester membrane.
Discontinue Cosmegen for burning or stinging sensation or other evidence indicating perivenous infiltration or extravasation.
Terminate the injection or infusion immediately and restart in another vein.
Intermittent application of ice to the site for 15 minutes 4 times daily for 3 days [see Warnings and Precautions ( 5.3)].
For injection: 500 mcg as a sterile, amorphous yellow to orange, lyophilized powder in a single-dose vial.
The risk of developing secondary malignancies, including leukemia, is increased following treatment with Cosmegen.
Severe and fatal hepatic veno-occlusive disease (VOD) can occur with Cosmegen. Risk factors for the development of VOD include age younger than 4 years or concomitant radiotherapy. After treatment with Cosmegen, monitor frequently for signs and symptoms of VOD; these include elevations in AST, ALT, total bilirubin, hepatomegaly, weight gain, or ascites. If patients develop VOD, considering delaying next dose of Cosmegen. Resume, reduce dose or permanently discontinue based on severity of reaction and disease being treated.
Extravasation of Cosmegen can result in severe local tissue injury manifesting as blistering, ulcerations and persistent pain requiring wide excision surgery followed by split-thickness skin grafting. If any signs or symptoms of extravasation occur, immediately interrupt the injection or infusion. Apply ice to the site intermittently for 15 minutes, 4 times a day for 3 days [see Dosage and Administration ( 2.7)] . Observe closely and consult plastic surgery if necessary based on severity of reaction.
Severe and fatal myelosuppression, which may include neutropenia, thrombocytopenia and anemia, can occur with Cosmegen. The nadir in neutrophil counts generally occurs 14 to 21 days after administration. Obtain complete blood counts prior to each treatment cycle. Delay next dose of Cosmegen if severe myelosuppression has not improved. Consider dose reduction for patients with prolonged myelosuppression based on severity of reaction and disease being treated.
The safety with live viral vaccines following Cosmegen has not been studied and vaccination with live virus vaccines is not recommended before or during treatment.
Severe mucocutaneous reactions, such as Steven-Johnson syndrome and Toxic Epidermal Necrolysis (TEN), can occur with Cosmegen. Permanently discontinue Cosmegen in patients who experience a severe mucocutaneous reaction.
Abnormalities of renal function can occur with Cosmegen. Monitor creatinine and electrolytes frequently during Cosmegen therapy.
Hepatotoxicity can occur with Cosmegen. Monitor AST, ALT, alkaline phosphatase, and bilirubin prior to and during Cosmegen therapy.
Cosmegen can increase radiation-induced gastrointestinal toxicity, myelosuppression, or erythema and vesiculation of the skin or buccal and pharyngeal mucosa. Reduce the dose of Cosmegen by 50% during concomitant radiation.
Radiation recall, affecting previously treated radiation fields, can occur in patients who receive Cosmegen after prior radiation therapy. Although the risk can occur with distant radiation exposure, the risk appears highest when Cosmegen is administered within two months of prior radiation.
Based on findings from animal studies and its mechanism of action, Cosmegen can cause fetal harm when administered to a pregnant woman. In animal reproduction studies, administration of dactinomycin to pregnant animals during the period of organogenesis was teratogenic, resulting in malformations at doses lower than the recommended human dose.
Advise pregnant women of the potential risk to the fetus. Advise females of reproductive potential to use effective contraception during treatment with Cosmegen and for at least 6 months after the final dose. Advise males with female partners of reproductive potential to use effective contraception during treatment with Cosmegen and for 3 months after the final dose [see Use in Specific Populations ( 8.1, 8.3)] .
Common adverse reactions are: infection, alopecia, rash, dysphagia, fatigue, fever, nausea, vomiting, anemia, neutropenia, thrombocytopenia, mucositis, and hepatotoxicity.
The following adverse reactions have been identified in clinical studies or postmarketing reports. Because some of these reactions were reported voluntarily from a population of uncertain size, it is not always possible to reliably estimate their frequency or establish a causal relationship to drug exposure.
Based on findings from animal studies and its mechanism of action, Cosmegen can cause fetal harm when administered to a pregnant woman [see Clinical Pharmacology ( 12.1)] . In animal reproduction studies, administration of dactinomycin to pregnant animals during the period of organogenesis was teratogenic, resulting in malformations at doses lower than the recommended human dose (see Data). Advise pregnant women of the potential risk to a fetus [see Use in Special Populations ( 8.3)] .
Dactinomycin was teratogenic in animals. Administration of dactinomycin to pregnant rats, rabbits, and hamsters during the period of organogenesis, increased the incidence of fetal malformations and caused embryotoxicity at doses (based on body surface area) as low as 0.2 times the clinical dose of 1250 mcg/m 2.
There are no data on the presence of dactinomycin or its metabolites in human milk or their effects on the breastfed infant or on milk production. Because of the potential for serious adverse reactions in breastfed infants from Cosmegen, advise women not to breastfeed during treatment with Cosmegen and, based on limited published data regarding the dactinomycin half-life, for 14 days after the final dose.
Verify the pregnancy status of females of reproductive potential prior to initiating Cosmegen [see Use in Specific Population ( 8.1)].
Cosmegen can cause fetal harm when administered to a pregnant woman [see Use in Specific Populations ( 8.1)] .
Advise females of reproductive potential to use effective contraception during treatment with Cosmegen and for at least 6 months after the final dose.
Because of the potential for genotoxicity, advise males with female partners of reproductive potential to use effective contraception during treatment with Cosmegen and for 3 months after the final dose [see Nonclinical Toxicology ( 13.1)] .
The safety and effectiveness of dactinomycin have been established in pediatric patients with Wilms tumor, rhabdomyosarcoma, Ewing sarcoma, and metastatic nonseminomatous testicular cancer .
The safety and effectiveness of dactinomycin have been established in post-menarchal pediatric patients with gestational trophoblastic neoplasia.
The safety and effectiveness of Cosmegen have not been established in pediatric patients undergoing regional perfusion for locally recurrent or locoregional solid malignancies.
Clinical studies of Cosmegen did not include sufficient numbers of subjects aged 65 and over to determine whether they respond differently from younger subjects.
Cosmegen (dactinomycin for injection) for intravenous use is a sterile, amorphous yellow to orange, lyophilized powder in a single-dose vial. Each vial contains 500 mcg of dactinomycin and 20 mg of mannitol.
Cosmegen is a cytotoxic actinomycin that binds DNA and inhibits RNA synthesis. The cytotoxic activity of dactinomycin has been demonstrated in animal models of different human cancers.
Dactinomycin exposure-response relationships and the time course of pharmacodynamics response are unknown.
The distribution and excretion of radioloabeled dactinomycin ( 3H actinomycin D) were assessed in three adult patients with malignant melanoma.
3H actinomycin D is concentrated in nucleated cells and does not penetrate the blood-brain barrier.
Following administration of radiolabeled dactinomycin, approximately 30% was recovered in urine and feces in one week.
Published studies and population analyses in patients ≤ 21 years of age with cancer report a trend of increasing systemic dactinomycin clearance with increasing body weight.
Published in vitro studies report that dactinomycin may be a substrate of the P-glycoprotein and OATP1B3 transporter systems.
Dactinomycin is a carcinogen in animals. Local sarcomas were produced in mice and rats after repeated subcutaneous or intraperitoneal injections. Mesenchymal tumors occurred in male rats given intraperitoneal injections of 50 mcg/kg, 2 to 5 times per week, for 18 weeks, at doses (based on body surface area) 0.5 times the clinical dose of 1250 mcg/m 2.
Dactinomycin was mutagenic in several in vitro and in vivo test systems including human fibroblasts and leukocytes, and HeLa cells. DNA damage and cytogenetic effects have been demonstrated in the mouse and the rat.
Cosmegen (dactinomycin for injection) for intravenous use is supplied as a sterile, amorphous yellow to orange, lyophilized powder in a single-dose vial. Each Cosmegen vial (NDC 55292-811-55) contains 0.5 mg of dactinomycin and 20 mg of mannitol.
Store at 20 to 25ºC (68 to 77ºF); excursions permitted between 15 to 30ºC (59 to 86ºF) [see USP Controlled Room Temperature].
Protect Cosmegen from light and humidity.
Store the reconstituted Cosmegen at room temperature for no more than 4 hours from reconstitution to completion of administration [see Dosage and Administration ( 2.7)] .
Advise patients of the increased risk of secondary malignancies [see Warnings and Precautions ( 5.1)] .
Advise patients about the symptoms of VOD and to seek medical attention if they develop new onset jaundice, abdominal distention, or right upper quadrant pain [see Warnings and Precautions ( 5.2)] .
Advise patients to contact their healthcare provider for any signs or symptoms of myelosuppression or infection [see Warnings and Precautions ( 5.4)] .
Advise patients of the risk of severe mucocutaneous reactions and to contact their health care provided for new skin lesions, mouth sores or oropharyngeal lesions [see Warnings and Precautions ( 5.5)] .
Advise patients of the need for periodic laboratory testing to monitor for renal toxicity and hepatotoxicity [see Warnings and Precautions ( 5.7, 5.8)] .
Advise patients of the risk of increased radiation-induced gastrointestinal, myelosuppression and skin toxicity [see Warnings and Precautions ( 5.9)] .
Advise females of reproductive potential of the potential risk to a fetus. Advise females to inform their healthcare provider of a known or suspected pregnancy [see Warnings and Precautions ( 5.10), Use in Specific Populations ( 8.1)] .
Advise females of reproductive potential to use effective contraception during treatment with Cosmegen and for 6 months after final dose [see Use in Specific Populations ( 8.3)] .
Advise male patients with female partners of reproductive potential to use effective contraception during treatment with Cosmegen and for 3 months after final dose [see Use in Specific Populations ( 8.3)] .
Advise females not to breastfeed during treatment with Cosmegen and for 14 days after the final dose [see Use in Specific Populations ( 8.2)] .
Manufactured for: Recordati Rare Diseases Inc., Lebanon, NJ 08833, U.S.A.
Cosmegen is a registered trademark of Recordati Rare Diseases Inc.
See USP controlled room temperature.
Protect from light and humidity.


text:
More fans to be allowed at Singapore Premier League matches from April
Fans at Hougang Stadium on Apr 24, 2021. (File photo: Singapore Premier League)
Ng Hong Siang
31 Mar 2022 04:59PM (Updated: 31 Mar 2022 04:59PM)
SINGAPORE: The number of spectators allowed at Singapore Premier League (SPL) matches will be raised to 75 per cent of the venue's capacity from Friday (Apr 1).
The revised limit is in line with the country's easing of COVID-19 measures that kicked in on Tuesday, the Football Association of Singapore (FAS) said in a news release on Thursday.
This announcement comes after the cap was raised to 50 per cent of each venue's capacity earlier in March.
Limits for the five SPL match venues for this season will be: 4,500 at Jalan Besar Stadium; 3,750 at Our Tampines Hub; and 2,250 each at Toa Payoh Stadium, Hougang Stadium and Jurong East Stadium.
While social distancing will not be required, spectators are encouraged to keep their masks on once inside the stadium.
Re-entry into the venues will be permitted, but spectators are required to be fully vaccinated to enter.
Children aged 12 and below, regardless of their vaccination status, must be accompanied by a fully vaccinated adult to gain entry, said FAS.
Food and drinks may be consumed, but only in specially designated areas in the stadiums, subject to prevailing dining-in requirements, which include eating and drinking at a fixed position, safe distancing and the prevailing group size limits of 10 people.
More fans allowed at Singapore Premier League matches from Mar 18
Upcoming Singapore Premier League season to have more matches
IN FOCUS: What does Singapore football need to raise its game?
Separately, FAS also announced the rescheduling of two Young Lions matches in April to allow them to go on local centralised training to prepare for the Southeast Asian Games in Hanoi.
The team was due to play Geylang International on Apr 23 and Tampines Rovers on Apr 27, but will now play these matches on Jun 21 and Aug 24 respectively.
Matchweek five of the SPL resumes on Friday, starting with a match between Hougang United and Albirex Niigata.
Source: CNA/nh(gr)
Football Association of Singapore


text:
WIRED, Contributor
Phone Companies Will Soon Banish Robocalls. For Real This Time
03/23/2017 02:19 pm ET Updated Mar 23, 2017
By Klint Finley for WIRED.
If Democrats, Republics, and the telecommunications industry can agree on anything, it's that robocalls are the worst. That's why the Federal Communications Commission passed rules today that expand the authority of phone companies to block unwanted phone calls. Short version: If a phone number seems at all likely to be bogus, companies can block it.
The law forbids marketers from calling people who are on the Do Not Call list. Yet according to the FCC, consumers last year received about 2.4 billion robocalls per month. And these calls are more than just an annoyance: Last year the IRS estimated that scammers posing as tax collectors used robocalls to make off with about $26.5 million from the public.
RELATED: The Senate Prepares to Send Internet Privacy Down a Black Hole
The FCC receives more complaints every month about robocalls than any other issue. But the agency struggles to track down culprits because most hide their real numbers using the same sorts of technology that make it possible to use a standard phone number with your Skype or Google Voice account. In most cases, the FCC says, robocallers don't even use real phone numbers. They rely on mimicking inactive or unassigned phone numbers — spoofing, in industry lingo.
With tracking down the culprits out of the question, phone companies have turned to automated blocking tools like Nomorobo, which blocks known robocallers while whitelisting emergency numbers. This practice is perfectly legal under FCC rules — in fact, the agency actively encourages carriers to provide customers with these types of tools. But some companies, including AT&T, complained that FCC rules against blocking calls prohibited them from aggressively targeting telemarketers for fear of accidentally blocking legitimate callers.
READ MORE: The FCC Head Says the Media Isn't the Enemy. In 2017, That's News
Today the FCC approved a proposal created in collaboration with a "strike force" that included AT&T, Apple, Google, and Comcast. Once the proposal's rules go into effect, carriers will have permission to block all calls from numbers that can't possibly be valid, such as those that aren't assigned to anyone or phone numbers that simply don't exist (like "000-000-0000").
"There is no reason why any legitimate caller should be spoofing an unassigned or invalid phone number," FCC chair Ajit Pai wrote in a blog post announcing the proposal. "It's just a way for scammers to evade the law."
It's possible scammers will move on to another technique once the proposal goes into effect, but for the time being at least your carrier can no longer blame the government for not blocking most robocalls.
More from WIRED:
9 Magical Photos Of California's Wildflower Super Bloom
Obama Talks AI and the Future of the World
America's Electronic Voting Machines Are Easy Targets
What Happens When You Talk About Salaries at Google
Hackers Trick Facial-Recognition Logins With Facebook Photos
A Hacking Group is Selling iPhone Spyware to Governments
CES FCC


BIRD METER IS NOT INCLUDED!!!!!! This one almost looks like brand new Preamp works (standby switch down) 600 plus watts pep Works great Fuse holder has been changed I do have a new factory fuse holder I can include. The item "Vintage Maco 300 Linear Tube Amplifier" is in sale since Monday, March 11, 2019. This item is in the category "Consumer Electronics\Radio Communication\Ham, Amateur Radio\Ham Radio Amplifiers".
The seller is "h_d87" and is located in Akron, New York. This item can be shipped to United States, Canada, United Kingdom, Denmark, Romania, Slovakia, Bulgaria, Czech republic, Finland, Hungary, Latvia, Lithuania, Malta, Estonia, Australia, Greece, Portugal, Cyprus, Slovenia, Japan, China, Sweden, South Korea, Indonesia, Taiwan, Belgium, France, Hong Kong, Ireland, Netherlands, Poland, Spain, Italy, Germany, Austria, Bahamas, Israel, New Zealand, Singapore, Switzerland, Norway, Saudi arabia, Ukraine, United arab emirates, Qatar, Kuwait, Bahrain, Croatia, Malaysia, Uruguay.


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