Microbial Science Research in the Post-COVID Environment.

Since early 2020, the world has witnessed the unprecedented accomplishments of the scientific community in the fight against the coronavirus disease 2019 (COVID-19) pandemic. In the meantime, we also learned valuable lessons and recognized the challenges that hindered our successes. In this article, we synthesize the ideas discussed at the ASM Virtual Symposium: Microbial Science Research in the Post-COVID Environment on 10 November 2020. We propose three new approaches that microbiology researchers can embrace to overcome these challenges. Moreover, we suggest broad systematic changes to focus on social impacts, teamwork, and diversity, equity, and inclusion. We believe these values are needed to prepare the microbial science research community for future opportunities and challenges.

Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth.

Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium Nitrosomonas europaea is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of N. europaea, e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on N. europaea have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on N. europaea Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene (nirK) was significantly lower. In contrast, both heme-copper-containing cytochrome c oxidases encoded by N. europaea were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of N. europaea's sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in N. europaea and other ammonia-oxidizing bacteria.IMPORTANCE Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, Nitrosomonas europaea, growing under oxygen-limited conditions.

Do Mid-Crown Enamel Formation Front Angles Reflect Factors Linked to the Pace of Primate Growth and Development?

Enamel formation front (EFF) angles represent the leading edge of enamel matrix secretion at particular points in time. These angles are influenced by rates of enamel extension (the rates at which tooth crowns grow in height), rates of enamel matrix secretion and the angles that prisms make with the enamel-dentine junction. Previous research suggests, but has not yet established, that these angles reflect aspects of primate biology related to their pace of growth and development, most notably brain and body size. The present study tested this possibility on histological sections using phylogenetically-controlled and Bonferroni-corrected analyses spanning a broad taxonomic range. Ten species were represented in the analysis of anterior teeth; 17 in the analysis of posterior (postcanine) teeth (with varying sample sizes). Also, tested was the relationship of EFF angles to striae of Retzius periodicity (long period growth rhythms in enamel) and degree of folivory, as both factors are related to primate developmental rates. Finally, several analyses were conducted to investigate whether tooth size (operationalized as EDJ length) might mediate these relationships. Central results are as follows: (1) Relationships between EFF angles and brain weight (anterior teeth) and between EFF angles and body mass (anterior and posterior teeth) are statistically significant and (2) Mid-crown EFF angles are not statistically significantly related to EDJ lengths. These results suggest that tooth size does not mediate relationships between EFF angles and brain weight/body mass and are discussed with respect to underlying enamel growth variables (especially rates of enamel extension and secretion). Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:125-139, 2018. © 2017 Wiley Periodicals, Inc.

Nitrobacter winogradskyi transcriptomic response to low and high ammonium concentrations.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2(-)) as a source of energy and nitrogen. In most natural situations, NO2(-) oxidation is coupled closely to ammonium (NH4(+)) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4(+) to meet its N-biosynthetic requirements. Interestingly, NH4(+) delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4(+) while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4(+) concentration. Genes in assimilatory NO2(-) metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4(+). Nitrobacter winogradskyi grown in medium with 25 mM NH4(+) upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4(+) and is likely to be modified during coupled nitrification with NH3 oxidizers.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Potential trends in Attention Deficit Hyperactivity Disorder (ADHD) drug use on a college campus: wastewater analysis of amphetamine and ritalinic acid.

Attention Deficit Hyperactivity Disorder (ADHD) medication use is on the rise in the United States. The most widely used ADHD medications are the amphetamine-type compounds Adderall (mixed amphetamine salts) and Ritalin (methylphenidate). According to survey data ADHD medications are used as a study drug or "Smart Drug" by students without a prescription on college campuses. Survey data of non-prescribed drug use has limitations with accurate reporting and no empirical data of usage exists in the literature. This study looks for trends in the use of these drugs on a college campus among low-stress and high stress periods. The metabolites of these two drugs, amphetamine and ritalinic acid, are quantified in campus wastewater using solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Trends show a possible increase in amphetamine levels during periods of high stress such as midterms, the last week of classes and finals week over levels from the baseline low stress weeks such as the first week of classes. Both semesters from the 2011-12 academic year were studied and the highest increase over baseline (760%) occurred during finals week of the second semester. Ritalinic acid levels gradually climbed first semester but had no obvious periodic trend second semester.

Hypoestrogenic "inactive phases" at the start of the menstrual cycle: changes with age and reproductive stage, and relationship to follicular depletion.

OBJECTIVE: To investigate hypoestrogenic "inactive phases" (IP) in the follicular phase of the menstrual cycle, with respect to age, reproductive stage, and follicular depletion. DESIGN: Analysis of prospectively collected menstrual bleed and estrone-3-glucuronide data. SETTING: Center for Population and Health, Georgetown University. PATIENT(S): White women (n = 88, aged 25-59 years, mean = 44.7 years) from the population-based Biodemographic Models of Reproductive Aging (BIMORA) project. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The IP durations by age and reproductive stage. Estimated follicular depletion rate based on IP durations. RESULT(S): Mean IP duration and variability decreased and then increased with age/reproductive stage. The proportion of very short (≤ 1 day) IP durations increased and then decreased with age/stage. Long IPs occurred most, but not exclusively, in the oldest age/latest stage. Follicular depletion rate estimates were a plausible 2%-4% per year of age, but these models were a poor fit because IP durations did not consistently increase across ages/stages. CONCLUSION(S): Follicular depletion models alone do not explain the observed pattern of IPs. Our data suggest that IPs reflect both follicular depletion and hyperstimulation in premenopausal and perimenopausal women. Knowledge of underlying IP patterns in the menstrual cycle could inform decisions about hormone sampling and contraception during the perimenopause.

Linear enamel hypoplasia as an indicator of physiological stress in great apes: reviewing the evidence in light of enamel growth variation.

Physiological stress, such as malnutrition or illness, can disrupt normal enamel growth, resulting in linear enamel hypoplasias (LEHs). Although ecological factors may contribute to LEH expression, other factors, such as surface abrasion and enamel growth variables, are also likely to be involved. Attention to these other factors is necessary before we can begin to understand what LEH might signify in terms of ecological sources of physiological stress in non-human primates. This study focuses on assessing the contribution of these other factors to variation in LEH expression within and across great ape taxa. Here, we present LEH data from unabraded crown regions in samples of seven great ape species. We analyze these data with respect to lateral enamel formation time and the angles that striae of Retzius make with the enamel surface, as these variables are expected to affect variation in LEH expression. We find that although the duration of enamel formation is associated with sex differences in LEH expression, it is not clearly related to taxonomic variation in LEH expression, and does not explain the low frequency of LEH in mountain gorillas found in this and a previous study. Our data on striae of Retzius angles suggest that these influence LEH expression along the tooth crown and may contribute to the consistently high frequencies of LEH seen in Pongo in this and previous studies. We suggest that future work aimed at understanding species variation in these angles is crucial to evaluating taxonomic patterns of LEH expression in great apes.

Conflict and death in a late prehistoric community in the American Midwest.

Over the past two decades, it has been recognized that the effects of intergroup conflict in prehistoric small-scale societies were greater than previously thought. Osteological evidence provides otherwise unobtainable information on the number of people who were killed, and who was most likely to become a casualty. One such site is Norris Farms #36 in the American Midwest, dating to ca. AD 1300. Skeletal evidence of injuries (blunt force trauma and arrow wounds), body mutilation (scalping, decapitation, and dismemberment), and scavenger damage indicate that one-third of the adults died in a series of ambushes, although children were mostly spared. Both young and old adults were killed, and the age distributions of the male and female victims were similar. Individuals with disabilities that interfered with mobility were more likely to be killed than their healthier counterparts. This level of conflict-related mortality almost certainly had an effect on the community's ability to conduct its affairs and, indeed, to survive as a viable economic and social group.

Longitudinal, epidemiologic studies of female reproductive aging.

Human female reproductive aging consists of multiple processes and interacts with other physiological systems in unique ways. Here we discuss eight recent longitudinal, epidemiologic studies of female reproductive aging that include endocrine data to highlight their contributions to our understanding of these various aging processes and their interactions. Specifically, we review data on ovarian and nonovarian reproductive aging processes and reproductive staging. We consider these data in the context of longitudinal research design and research goals, identify limitations of the studies but also ways in which existing longitudinal data can further our understanding of aging processes, and make recommendations for future studies of female reproductive aging.

Progesterone and ovulation across stages of the transition to menopause.

OBJECTIVE: Detailed characterization of progesterone and ovulation across the menopausal transition provides insight into conception risk and mechanisms of reproductive aging. METHODS: Participants (n = 108, aged 25-58 y) collected daily urine specimens for 6-month intervals in each of 5 consecutive years. Specimens were assayed for pregnanediol glucuronide (PDG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrone glucuronide (E1G). Reproductive stage was determined using cycle length variance. A hierarchical algorithm was used to identify ovulation. Linear mixed-effects models estimated (1) the frequency and day of ovulation by age and stage; (2) differences in FSH, LH, and E1G levels between ovulatory (O) and anovulatory (AO) cycles; and (3) total PDG levels and PDG levels in O cycles by age and stage. RESULTS: The probability of AO cycles increased across the perimenopause (P < 0.0001); reproductive stage was a stronger predictor than age of the probability of anovulation. Most cycles in late perimenopause were AO (>60%), but one quarter of cycles longer than 60 days were O. Average day of ovulation was later in the late perimenopause (mean [SD] cycle day, 27 [25] d) compared with the premenopause. FSH and LH levels were higher and E1G levels were lower in AO than O cycles (P < 0.0001 for each). Total PDG decreased in the late perimenopause, but 95th percentile PDG in O cycles declined steadily across the transition. CONCLUSIONS: Exposure to the risk of conception in women experiencing cycles long enough to classify them as late perimenopausal is far from negligible. Reproductive stage is more informative than age about PDG levels and the likelihood of anovulation.

Sex differences in anthropoid mandibular canine lateral enamel formation.

Previous research has demonstrated that great ape and macaque males achieve large canine crown sizes primarily through extended canine growth periods. Recent work has suggested, however, that platyrrhine males may achieve larger canine sizes by accelerating rather than prolonging growth. This study tested the hypothesis that the ontogenetic pathway leading to canine sexual dimorphism in catarrhines differs from that of platyrrhines. To test this hypothesis, males and females of several catarrhine genera (Hylobates, Papio, Macaca, Cercopithecus, and Cercocebus) and three platyrrhine genera (Cebus, Ateles, and Callicebus) were compared in the number and spacing of perikymata (enamel growth increments) on their canine crowns. In addition, perikymata periodicities (the number of days of growth perikymata represent) were determined for five genera (Hylobates, Papio, Macaca, Cebus, and Ateles) using previously published as well as original data gathered for this study. The central findings are as follows: 1) males have more perikymata than females for seven of eight genera (in five of the seven, the differences are statistically significant); 2) in general, the greater the degree of sexual dimorphism, the greater the sex difference in male and female perikymata numbers; 3) there is no evidence of a systematic sex difference in primate periodicities; and 4) there is some evidence that sex differences in enamel formation rates may make a minor contribution to canine sexual dimorphism in Papio and Cercopithecus. These findings strongly suggest that in both catarrhines and platyrrhines prolongation of male canine growth is the primary mechanism by which canine crown sexual dimorphism is achieved.

Total and unopposed estrogen exposure across stages of the transition to menopause.

Detailed characterization of estrogen dynamics during the transition to menopause is an important step toward understanding its potential implications for reproductive cancers developing in the transition years. We conducted a 5-year prospective study of endogenous levels of total and unopposed estrogen. Participants (n=108; ages 25-58 years) collected daily urine specimens for 6 months in each of 5 consecutive years. Specimens were assayed for estrone-3-glucuronide (E1G) and pregnanediol-3-glucuronide. Linear mixed-effects models were used to estimate exposure to total and unopposed estrogen by age and reproductive stage. Reproductive stage was estimated using menstrual cycle length variance. E1G mean area under the curve and mean E1G 5th and 95th percentiles represented total estrogen exposure. An algorithm identifying days of above-baseline E1G that coincided with the days of baseline pregnanediol-3-glucuronide was used to identify days of unopposed estrogen. Mean E1G area under the curve increased with age in the pretransition and early transition and decreased in the late transition. Ninety-fifth percentile E1G levels did not decline until after menopause, whereas 5th percentile levels declined from the early transition to the postmenopause. The number of days of unopposed estrogen was significantly higher during the transition compared with the pretransition. Given the length of time women spend in the transition, they are exposed to more total and unopposed estrogen than has been previously appreciated. Coupled with epidemiologic evidence on lifetime exposure to estrogen, these results suggest that variation in the amount of time spent in the transition may be an important risk factor for reproductive cancers.

Monitoring reproductive aging in a 5-year prospective study: aggregate and individual changes in luteinizing hormone and follicle-stimulating hormone with age.

OBJECTIVE: This study describes age-related changes in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a 5-year prospective study of reproductive aging. DESIGN: Participants (n = 156 college-educated, white, US women; 25 to 58 y) were recruited from the TREMIN Research Program on Women's Health. They collected daily urine specimens for 6 months in each of 5 consecutive years. Specimens were assayed for LH and FSH. Aggregate changes were calculated in LH and FSH with age, and multilevel models were used to estimate individual hormone trajectories and within-woman and between-woman variances by age. RESULTS: Aggregate LH levels increased beginning after age 45; FSH increased at all ages, accelerating after age 45. Individual-level patterns with age included the following: reproductive-age LH and FSH levels, with increasing FSH and increasing or decreasing LH (ages 20 to 49); rapidly increasing LH and FSH (ages 40 to 59); and increasing or steady postmenopausal LH and FSH (ages 46 to 62). FSH levels were consistently high in the latter category, but LH levels overlapped with levels found in younger women (<45 y). Individual LH patterns showed more variability (5% to 35% of total variance) than FSH (3% to 22% of total variance). Both hormones had relatively low variation within individuals compared with between-woman differences (65% to 97% of total variance). CONCLUSIONS: Aggregate-level data do not reflect differences across women and oversimplify the age-related increases and variability in LH and FSH. Individual FSH levels are not distinguishable from reproductive-age levels until after rapid perimenopausal increases in FSH occur; individuals vary in whether their postmenopausal LH levels are distinguishable from reproductive-age levels.

The length of perimenopausal menstrual cycles increases later and to a greater degree than previously reported.

OBJECTIVE: To demonstrate that the perimenopausal increase in menstrual cycle length presented by Treloar et al. was biased by misidentified menopause dates, mean values classified by calendar year, and exclusion of menstrual cycles straddling two calendar years; and to use the revised data to investigate women's experiences of longer perimenopausal cycles. DESIGN: Secondary analysis of prospectively collected menstrual cycle data. SETTING: Center for Population and Health, Georgetown University. PATIENT(S): One hundred twenty white, college-educated, US women in the Tremin Research Program on Women's Health. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Mean cycle length and time spent in >40-day cycles, by year before menopause. RESULT(S): Mean estimates for each of the 4 years before menopause were 30.48, 35.02, 45.15, and 80.22 days, respectively, compared with the original analysis: 33.60, 43.91, 55.87, and 54.58 days. In the year before menopause, the majority of women spent >or=75% of their time in cycles >40 days long. CONCLUSION(S): Treloar's estimates of mean cycle length were biased. Long cycles occurred throughout perimenopause, but the largest increase in mean cycle length did not occur until the final year before menopause. New estimates of the time spent in cycles >40 days may be useful clinically as well as epidemiologically for assessing menopausal onset and symptomatology.

The relationship between number of striae of Retzius and their periodicity in imbricational enamel formation.

Imbricational crown formation times (ICFTs) estimated from the number of perikymata on tooth surfaces are error-prone because the number of days between adjacent perikymata varies across individuals and species, and is only visible within tooth microstructure. We investigated striae of Retzius (SR) numbers (analogous to perikymata numbers), SR periodicities (days between SR or perikymata), and ICFTs for a mandibular canine sample (n=49) from medieval Denmark. We tested the relationship between SR number and periodicity to determine whether regression formulae could be produced that would allow periodicity (and ICFTs) to be determined from surface perikymata numbers. Periodicities (range=7-11 days, mode=8) and SR numbers (range=142-257, mean=190.3, s.d.=27.5) were normally distributed; ICFTs were non-normal (mean=1,594 days, s.d.=65.7). We tested periodicity as a quadratic, linear, and log-log transform linear function of SR number and found an inverse relationship (quadratic: R2=0.9504; linear: R2=0.9138; log-log transform: R2=0.9418; all p<0.001) that allowed estimation of periodicity from SR or perikymata numbers in this population and tooth type. If periodicity and SR number are inversely related in other hominin taxa, studies that have estimated ICFT by multiplying perikymata number by a human modal periodicity value or made inferences about development based only on perikymata numbers may have introduced substantial error into their ICFT estimates and life history inferences. The inverse relationship is similar to that predicted by a model of SR formation in which the ICFT for a given tooth type and population is held constant and all combinations of periodicity and SR number result in the same ICFT. However, we found that lower periodicities had longer ICFTs and higher periodicities had shorter ICFTs than the model predicted, suggesting that the model may not reflect the real process, or that there are other factors (e.g., sample size, misclassification, sexual dimorphism) also affecting the relationship between periodicity and SR number.

Monitoring reproductive aging in a 5-year prospective study: aggregate and individual changes in steroid hormones and menstrual cycle lengths with age.

OBJECTIVE: We describe a 5-year prospective study of reproductive aging, and present analyses of steroid hormone and menstrual cycle changes with age. DESIGN: Participants were college-educated white women, primarily of northern European ancestry, recruited from the Tremin Research Program on Women's Health (n = 156, 25-58 years). In each of 5 consecutive years, they collected daily urine specimens for 6 months and recorded menstrual bleeds for all months. Urine specimens were assayed for estrone-3-glucuronide (E1G) and pregnanediol-3-glucuronide (PDG), urinary metabolites of estradiol and progesterone. Using multilevel models, we estimated hormone and cycle-length trajectories for individual women and within- and between-woman variance by age. RESULTS: At the aggregate level, PDG declined beginning in the 30s, E1G increased into the 40s before declining, and cycle length became more variable with age. Individual-level models revealed substantial hormonal variation across women, in both absolute levels and rates of change. Most women showed declining E1G by the late 40s, declining PDG in the 30s, and increasing mean cycle length in the 40s. Hormonal variation decreased with age; cycle length variation decreased and then increased. Within individual women, cycle lengths were highly variable while hormone levels were more stable. Women differed more from each other in hormone levels than for cycle lengths. CONCLUSIONS: Aggregate-level analyses show general changes in steroid hormones and cycle length but cannot show variation within and across women. Individuals' cycle lengths were too variable to predict hormone levels. Clinicians should obtain more data on individual women's hormonal patterns when determining fertility or menopause treatments.

The temporal dynamics of the fourteenth-century Black Death: new evidence from English ecclesiastical records.

Recent research has questioned whether the European Black Death of 1347-1351 could possibly have been caused by the bubonic plague bacillus Yersinia pestis, as has been assumed for over a century. Central to the arguments both for and against involvement of Y. pestis has been a comparison of the temporal dynamics observed in confirmed outbreaks of bubonic plague in early-20th-century India, versus those reconstructed for the Black Death from English church records--specifically, from lists of institutions (appointments) to vacated benefices contained in surviving bishops' registers. This comparison is, however, based on a statistical error arising from the fact that most of the bishops' registers give only the dates of institution and not the dates of death. Failure to correct for a distributed (as opposed to constant) lag time from death to institution has made it look as if the Black Death passed slowly through specific localities. This error is compounded by a failure to disaggregate the information from the bishops' registers to a geographical level that is genuinely comparable to the modern data. A sample of 235 deaths from the bishop's register of Coventry and Lichfield, the only English register to list both date of death and date of institution, shows that the Black Death swept through local areas much more rapidly than has previously been thought. This finding is consistent with those of earlier studies showing that the Black Death spread too rapidly between locales to have been a zoonosis such as bubonic plague. A further analysis of the determinants of the lag between death and institution, designed to provide a basis for reexamining other bishops' registers that do not provide information on date of death, shows that the distribution of lags could vary significantly by time and space even during a single epidemic outbreak.