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When vertebrates are exposed to stressors, the subsequent acute increase in glucocorticoids by the hypothalamic-pituitaryadrenal (HPA) axis triggers a suite of adaptive responses, including mobilization of stored energy and repression of non-essential processes. However, chronic exposure to high concentrations of glucocorticoids can lead to metabolic dysregulation, impaired immune function, and cognitive decline. In developing young, this hormonal stress response shows considerable variation. Generally, the physiological stress response of young of precocial species is comparable to that of adults, whereas offspring of altricial species exhibit an attenuated response compared to adults. The developmental hypothesis of the HPA axis proposes that the dampened stress response in dependent offspring is an adaptive response to avoid the negative effects of elevated glucocorticoids, particularly in altricial species where young lack the ability to mitigate stressful stimuli.We aimed to test the developmental hypothesis in a tropical avian species, the lance-tailed manakin (Chiroxiphia lanceolata). We predicted that nestlings of this altricial species should have a dampened corticosterone response, in both magnitude and duration, compared to that of adults. We also predicted that recently fledged hatch-year birds would display a response intermediate to that of adults and nestlings. We quantified circulating corticosterone levels in adults, recently fledged hatch-year birds, and 11-day-old nestlings using a standardized capture and restraint protocol. Nestlings showed a lower maximal corticosterone response and faster negative feedback compared to adults. Further, five post-fledging hatch-year birds showed a feedback response intermediate to those of nestlings and adults. However, we caution against generalizing about fledgling responses beyond this study due to the small sample (n = 5). Interestingly, lance-tailed manakin nestlings appear to return to baseline concentrations faster than nestlings of temperate species. These results support the developmental hypothesis of the HPA axis explaining variation in stress response. This study is the first to assess the development of the hormonal stress response in nestlings of a tropical bird, which is of interest because of our still-developing understanding of how tropical and temperate species differ physiologically. Finally, findings here underscore the importance of validating and adjusting sampling protocols that quantify nestling stress responses, as sampling timelines identified for adults may underestimate the magnitude of the nestling stress response., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glucocorticoids are steroid hormones which increase dramatically in response to a physical or perceived stressor. However, developing young of altricial species typically have a damped glucocorticoid stress response. The developmental hypothesis posits that the physiological stress response should develop concurrently with an individual's ability to respond to a challenge. The dampened response may benefit an organism, as chronic exposure to glucocorticoids can have short- and long-term detrimental effects, and altricial young are unable to escape most stressors. However, we do not know if or why species with similar ontogeny vary in their development of the physiological stress response. We assessed levels of baseline and stress-induced corticosterone (the main avian glucocorticoid) in six passerine species with varying life-history strategies, including a brood parasite, the brown-headed cowbird (Molothrus ater). Circulating baseline corticosterone levels increased with nestling age for all species. Stress-induced corticosterone levels sampled at 15-min post-capture significantly increased with nestling age at a similar rate and magnitude in brown-headed cowbirds, eastern phoebes (Sayornis phoebe), hooded warblers (Setophaga citrina), red-winged blackbirds (Agelaius phoeniceus), and song sparrows (Melospiza melodia). However, gray catbird (Dumetella carolinensis) nestlings showed an extremely dampened elevation in corticosterone in response to stress, even near fledge. Gray catbirds are unusual among songbirds, as they are open-ended song learners. Stress during development can profoundly influence avian song learning and performance abilities. However, further study is necessary to determine if there is a relationship between nestling adrenocortical activity and open- versus closed-ended song learning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The El Niño Southern Oscillation (ENSO) is a global phenomenon that influences climate variation and, in turn, the ecological processes affecting the abundance and distribution of populations across taxa. For example, the ENSO can profoundly influence the development and survival of pelagic species, but the extent to which the ENSO affects offspring of terrestrial species is less well known. We used piecewise structural equation modeling to investigate the direct and indirect relationship between the ENSO and offspring development and survival in a terrestrial tropical passerine, the lance-tailed manakin (Chiroxiphia lanceolata). The Oceanic Niño Index (ONI), a measure of the ENSO, was negatively related to individual growth rate, maximum number of lesion developed by nestlings, and hatching day-of-year; which in turn mediated indirect effects on fledging success and recruitment. Further the ONI was a better predictor of nestling development compared to local temperature and rainfall. Our study establishes a link between the ENSO and the development and survival of young of a terrestrial species and underscores the need to better understand how offspring cope with global climate variation.

A process evaluation of nurses' implementation of an infant-feeding counseling protocol was conducted for the Breastfeeding, Antiretroviral and Nutrition (BAN) Study, a prevention of mother-to-child transmission of HIV clinical trial in Lilongwe, Malawi. Six trained nurses counseled HIV-infected mothers to exclusively breastfeed for 24 weeks postpartum and to stop breastfeeding within an additional four weeks. Implementation data were collected via direct observations of 123 infant feeding counseling sessions (30 antenatal and 93 postnatal) and interviews with each nurse. Analysis included calculating a percent adherence to checklists and conducting a content analysis for the observation and interview data. Nurses were implementing the protocol at an average adherence level of 90% or above. Although not detailed in the protocol, nurses appropriately counseled mothers on their actual or intended formula milk usage after weaning. Results indicate that nurses implemented the protocol as designed. Results will help to interpret the BAN Study's outcomes.

Objective: To learn the attitudes and concerns of the local community on participating in research, infant feeding practices, and maternal nutrition in order to inform the design of a clinical trial in Lilongwe, Malawi on the safety and efficacy of antiretroviral and nutrition interventions to reduce postnatal transmission of HIV., Design: Formative research methods were used, including semi-structured interviews, focus group discussions, home observations, and taste trials. Data were collected, analyzed, and incorporated into the protocol within 3 months., Results: Participants were supportive of the clinical trial, although their overall understanding of research was limited. Mothers agreed that infants' blood could be drawn by venipuncture, yet concern was raised about the amount of blood proposed to be collected from both infants and mothers. Data demonstrated that rapid breastfeeding cessation would be difficult and malnutrition could be a risk if infants were weaned early. Mothers selected a maternal supplement suitable for use in the clinical trial., Conclusions: The protocol was rapidly modified to achieve cultural acceptability while maintaining study objectives. Without the formative research, several significant areas would have been undetected and may have jeopardized the implementation of the trial. Additional research was carried out to develop a meaningful informed consent process, the amount of blood collected was reduced to acceptable levels, and the protocol was modified to reduce the risk of malnutrition. Researchers who conduct clinical trials are encouraged to incorporate formative research into their protocol design to ensure participant understanding of the research, to safeguard participants, and to increase feasibility and acceptance of the clinical research in the community.

Background: Low micronutrient levels observed with increasing adiposity may result from inadequate nutrient intake and/or alterations in nutrient metabolism., Objective: To examine the association between body mass index (BMI) and micronutrient levels among a nationally representative sample of US adults aged > or = 19 years., Design: Using nationally representative cross-sectional data from the National Health and Nutrition Examination Survey III (NHANES III), we examined odds ratios of low micronutrient levels using logistic regression adjusting for covariates., Measurements: Nutritional biomarker levels (as indicated by serum levels of total carotenoids, alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein/zeaxanthin, lycopene, vitamin E, vitamin C, selenium, vitamin A, vitamin D, folate, vitamin B12, and red blood cell folate) among men and nonpregnant women, by BMI category., Results: Overweight and obese adults had higher odds of low levels for a number of nutrients compared with normal-weight adults. Odds of being low in multiple micronutrients was most common among overweight and obese premenopausal women., Conclusion: These findings underscore the need for further assessment of specific micronutrient inadequacies among persons who are overweight or obese. Specifically, research is needed to determine whether these inadequacies are due to insufficient dietary intake, altered metabolic processes, or both.

Context: There are scant data on patterns of multivitamin use among US adults in terms of body mass index (BMI) or whether one is trying to lose weight., Objective: To examine multivitamin use and beliefs about multivitamin use among adults according to BMI and to determine whether use by body weight differs if one is trying to lose weight., Design: Cross-sectional multivariate analysis of the HealthStyles consumer survey. The final analytic sample consisted of 2239 women and 1532 men., Main Outcome Measures: Prevalence and odds of multivitamin use by demographic and behavioral characteristics including BMI, use by weight loss intent, and among users, reasons for use., Results: 63.7% of women and 52.9% of men reported multivitamin use (taking 1 or more multivitamin per week). Obese women were less likely than normal-weight women to use multivitamins; no differences according to BMI category were detected for men. Among women who were not trying to lose weight, obese women were less likely than normal-weight women to use multivitamins (odds ratio = 0.63, CI 0.41-0.98). Assessment of reasons for use found that compared among women not trying to lose weight, those trying to lose weight were more likely to report multivitamin use because It is important for my health., Conclusions: This descriptive analysis adds to the limited literature on multivitamin use according to both body weight and attempting to lose weight. Multivitamin use was common and decreased with increasing BMI. This may be because fewer obese people consider vitamins "important for their health".

Background: To prevent postnatal transmission of HIV in settings where safe alternatives to breastfeeding are unavailable, the World Health Organization (WHO) recommends exclusive breastfeeding followed by early, rapid cessation of breastfeeding. Only limited data are available on the attitudes of health workers toward this recommendation and the impact of these attitudes on infant feeding counselling messages given to mothers., Methods: As part of the Breastfeeding, Antiretroviral, and Nutrition (BAN) clinical trial, we carried out an in-depth qualitative study of the attitudes, beliefs, and counselling messages of 19 health workers in Lilongwe, Malawi., Results: Although none of the workers had received formal training, several reported having counseled HIV-positive mothers about infant feeding. Health workers with counselling experience believed that HIV-infected mothers should breastfeed exclusively, rather than infant formula feed, citing poverty as the primary reason. Because of high levels of malnutrition, all the workers had concerns about early cessation of breastfeeding., Conclusion: Important differences were observed between the WHO recommendations and the attitudes and practices of the health workers. Understanding these differences is important for designing effective interventions.

A neglected issue in the literature on maternal nutrition and HIV is how HIV-positive women perceive their own bodies, health, and well-being, particularly in light of their infection, and whether these perceptions influence their infant feeding practices and their perceived ability to breast-feed exclusively through 6 mo. We conducted formative research to better understand breast-feeding practices and perceptions, and to inform the Breastfeeding, Antiretroviral, and Nutrition (BAN) Study, a clinical trial to evaluate antiretroviral and nutrition interventions to reduce mother-to-child transmission of HIV during breast-feeding in Lilongwe, Malawi. Twenty-two HIV-positive women living in semi-rural areas on the periphery of Lilongwe participated in in-depth interviews. In an adaptation of the body-silhouette methodology, nine culturally appropriate body silhouettes, representing a continuum of very thin to very large shapes, were used to elicit women's views on their present, previous-year, and preferred body shapes, and on the shape they perceived as healthy. The narrative scenario method was also used to explore women's views on 2 fictional women infected with HIV and their ability to exclusively breast-feed. Women perceived larger body shapes as healthy, because fatness is considered a sign of good health and absence of disease, and many recognized the role of nutrition in achieving a preferred or healthy body shape. Several women believed their nutritional status (body size) was declining because of their illness. Women were concerned that breast-feeding may increase the progression of HIV, suggesting that international guidelines to promote appropriate infant feeding practices for infants whose mothers are infected with HIV should focus on the mother's health and well-being, as well as the infant's.

Clinical evidence shows that combining advice to increase fruit and vegetable consumption with caloric restriction is an effective strategy for weight management. The purpose of this review is to evaluate epidemiologic evidence to determine whether it supports an association between fruit and/or vegetable consumption and body weight. Few studies have been designed to specifically address this issue, and those that are available vary in methodology and offer inconsistent results. We make recommendations on how to strengthen future studies so that the influence of fruit and vegetable consumption on body weight in free-living individuals is better understood.

Given the recent surge in obesity, effective dietary strategies for weight management are required. Because fruits and vegetables are high in water and fiber, incorporating them in the diet can reduce energy density, promote satiety, and decrease energy intake. Although few interventions have specifically addressed fruit and vegetable consumption, evidence suggests that coupling advice to increase intake of these foods with advice to decrease energy intake is a particularly effective strategy for weight management. This approach may facilitate weight loss because it emphasizes positive messages rather than negative, restrictive messages.

A transformation system was developed for Bacillus megaterium by using antibiotic resistance plasmid deoxyribonucleic acid molecules derived from Staphylococcus aureus and Bacillus cereus. Lysozyme-generated protoplasts of B. megaterium allowed uptake of plasmid deoxyribonucleic acid in the presence of polyethylene glycol. Transformants expressed the antibiotic resistance determinants present on the plasmid deoxyribonucleic acid, and reisolated plasmid deoxyribonucleic acid yielded restriction endonuclease digestion patterns identical to those of the donor deoxyribonucleic acid.

A host-vector system was developed for molecular cloning in Bacillus megaterium and used to clone the structural and immunity genes for megacins A-216 and A-19213. Recombinant clones that expressed immunity only or both immunity to and production of each megacin were obtained. Restriction mapping of native megacinogenic plasmids and recombinant clones was used to construct physical and genetic maps of megacinogenic plasmids pBM309 and pBM113. Limited sequence homology between pBM309 and pBM113 was detected by Southern blot hybridization and was mapped to, at most, a 6.4-kilobase-pair region of pBM309 and a 6.1-kilobase-pair region of pBM113.

The gene encoding the 66-kDa entomocidal protein (P2 protein or mosquito factor) from Bacillus thuringiensis var. kurstaki has been isolated by the use of a 62-mer oligonucleotide probe that encoded 21 amino acids of the P2 protein NH2 terminus. The DNA sequence of the gene, designated cryBI, was unique from the published sequences of other B. thuringiensis genes. However, the amino acid sequence of the P2 protein, as deduced from the DNA sequence of the cryBI gene, was found to contain a sequence of 100 amino acids having 37% homology to a group of B. thuringiensis entomocidal proteins, the P1 proteins. Late stationary phase Bacillus megaterium cells harboring the cloned B. thuringiensis cryBI gene contained large aggregates of the P2 protein, and the cells were highly toxic to both lepidopteran and dipteran larvae. In contrast, Escherichia coli cells harboring the cloned cryBI gene contained very low levels of the P2 protein. DNA blot hybridization experiments showed that certain B. thuringiensis strains contained at least one cryBI-related DNA sequence in addition to the cryBI gene itself.

Megacins A-216 and A-19213 in Bacillus megaterium are plasmid encoded, as shown by analysis of cured, non-megacinogenic (Meg-) derivatives of strains 216 and ATCC 19213 and by polyethylene glycol-mediated protoplast transformation of Meg- bacteria with plasmid DNA. The results of both techniques implicated a 31-megadalton plasmid, pBM309, in megacin A-216 production and a 29-megadalton plasmid, pBM113, in megacin A-19213 production.

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of approximately 10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry-) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry- BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry+ and Cry- variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry+ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.

The circular deoxyribonucleic acid (DNA) of Bacillus megaterium was fractionated by sedimentation velocity on preparative zonal gradients. The fractions obtained were characterized by analytical sedimentation velocity analysis on neutral and alkaline sucrose gradients, and by contour length measurement by electron microscopy. Each fraction was found to contain circular molecules of one to three discrete sizes, in various combinations of covalently closed and open circular forms. Estimations of the molecular masses of these sizes gave values of 3.9, 6.2, 16.0, 31, and 60 million daltons for the major molecular species. Minor amounts of molecules of 7.6, 11.7, 47, 89, and 112 million daltons were observed in the electron microscope analyses. Length measurements of almost 600 molecules from the various fractions showed that all except six could be placed in distinct size classes. The distribution of molecular sizes in unfractionated circular DNA was shown to consist primarily of the two smallest size classes, although the relative proportions by weight of five of the classes were roughly equivalent.

The recently discovered high-frequency transfer of plasmids between strains of Bacillus thuringiensis was used to study the genetic relationship between plasmids and production of the insecticidal delta-endotoxin crystal. Three strains of B. thuringiensis transmitted the Cry+ (crystal-producing) phenotype to Cry- (acrystalliferous) B. thuringiensis recipients. Agarose gel electrophoresis showed that one specific plasmid from each donor strain was always present in Cry+ "transcipients." The size of the transmissible crystal-coding plasmid varied with the donor strain, being 75 MDal (megadaltons) in size in HD-2, 50 MDal in HD-73, and 44 MDal in HD-263. Immunological analysis showed the Cry+ transcipients to be hybrid strains, having flagella of the recipient serotype and crystals of the donor serotype. These results demonstrate that the structural genes for the delta-endotoxin are plasmid borne. Crystal-coding plasmids also transferred into two strains of the related species Bacillus cereus and yielded transcipients that produced crystals of the same antigenicity as the donor strain.

The polydisperse circular deoxyribonucleic acid (DNA) molecules which comprise up to 30% of the total extractable DNA of Bacillus megaterium strain 216 have been purified and partially characterized. Banding in cesium chlorideethidium bromide by "gradient relaxation" in a fixed-angle rotor provided good resolution of circular and chromosomal DNAs for preparative separations. Renaturation studies on purified circular DNA failed to reveal a rapidly renaturing fraction, and DNA-DNA hybridization studies indicated that the majority of the chromosomal nucleotide sequences are represented in the heterogeneous-size population of circular molecules. It is concluded that the circular DNA of B. megaterium does not represent typical bacterial plasmid DNA. The possibility that the circular DNA molecules are the result of the expression of a defective bacteriophage is discussed.

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.

Forty tryptophan auxotrophs of Bacillus subtilis have been placed in six phenotypic classes on the basis of growth responses, accumulation properties, and, in some cases, specific enzymatic defects. Three-point transformation crosses between representative mutants of the six different types have permitted the determination of the orders of the gene loci. In addition, mutational site orders for mutants within each of the classes have been determined by the same techniques. The organization of the cluster of genes controlling tryptophan biosynthesis in B. subtilis appears to be essentially analogous to that of Escherichia coli and Salmonella typhimurium.

Carlton, Bruce C. (Yale University, New Haven, Conn.). Fine-structure mapping by transformation in the tryptophan region of Bacillus subtilis. J. Bacteriol. 91:1795-1803. 1966.-The arrangements of eight mutational sites within a segment of the tryptophan cluster of Bacillus subtilis have been determined by three-point reciprocal crosses with a mutation in the same gene cluster used as an outside marker. Estimates of distances between sites were made by two-point crosses and normalization to recombination values for an unlinked marker to correct for physiological variabilities between recipient strains. In general, the additivities of map distances determined by this procedure were reasonable, although some discrepancies were noted in reciprocal crosses between widely separated sites within the gene region examined. No definitive polarity effect on recombination was evident, and it appears that the variations observed in reciprocal crosses may be an effect of the probability of recovery of crossovers between exogenotic and endogenotic segments. It is concluded that fine-structure mapping is feasible for the transformation system in this organism and that a high degree of resolution can be attained.

Two protease-deficient mutants of Bacillus subtilis 168 (Trp(-)) were isolated and compared with the parental strain with respect to production of intracellular proteases and sporulation. A mutant lacking the metal-requiring "neutral" protease intracellularly sporulated as well as the parental strain. A second mutant, deficient in an as yet uncharacterized intracellular protease, failed to sporulate normally. It is proposed that this new protease is also involved in intracellular protein turnover.

DNA preparations from two strains of Bacillus megaterium, one which produces megacin and another that is megacin sensitive, were analyzed by dye-buoyant density centrifugation, cesium chloride gradient equilibrium centrifugation in the absence of dye, zone sedimentation in sucrose, and electron microscopy. Both strains were found to contain a substantial proportion of their total DNA as circular, covalently closed, double helical molecules. These DNA elements were heterogeneous in size and appeared slightly less dense than chromosomal DNA. The physiological role and origin of these elements is unknown at present.