Deconstructing sex: Strategies for undoing binary thinking in neuroendocrinology and behavior.

The scientific community widely recognizes that "sex" is a complex category composed of multiple physiologies. Yet in practice, basic scientific research often treats "sex" as a single, internally consistent, and often binary variable. This practice occludes important physiological factors and processes, and thus limits the scientific value of our findings. In human-oriented biomedical research, the use of simplistic (and often binary) models of sex ignores the existence of intersex, trans, non-binary, and gender non-conforming people and contributes to a medical paradigm that neglects their needs and interests. More broadly, our collective reliance on these models legitimizes a false paradigm of human biology that undergirds harmful medical practices and anti-trans political movements. Herein, we continue the conversations begun at the SBN 2022 Symposium on Hormones and Trans Health, providing guiding questions to help scientists deconstruct and rethink the use of "sex" across the stages of the scientific method. We offer these as a step toward a scientific paradigm that more accurately recognizes and represents sexed physiologies as multiple, interacting, variable, and unbounded by gendered preconceptions. We hope this paper will serve as a useful resource for scientists who seek a new paradigm for researching and understanding sexed physiologies that improves our science, widens the applicability of our findings, and deters the misuse of our research against marginalized groups.

Systematic investigation of imprinted gene expression and enrichment in the mouse brain explored at single-cell resolution.

BACKGROUND: Although a number of imprinted genes are known to be highly expressed in the brain, and in certain brain regions in particular, whether they are truly over-represented in the brain has never been formally tested. Using thirteen single-cell RNA sequencing datasets we systematically investigated imprinted gene over-representation at the organ, brain region, and cell-specific levels. RESULTS: We established that imprinted genes are indeed over-represented in the adult brain, and in neurons particularly compared to other brain cell-types. We then examined brain-wide datasets to test enrichment within distinct brain regions and neuron subpopulations and demonstrated over-representation of imprinted genes in the hypothalamus, ventral midbrain, pons and medulla. Finally, using datasets focusing on these regions of enrichment, we identified hypothalamic neuroendocrine populations and the monoaminergic hindbrain neurons as specific hotspots of imprinted gene expression. CONCLUSIONS: These analyses provide the first robust assessment of the neural systems on which imprinted genes converge. Moreover, the unbiased approach, with each analysis informed by the findings of the previous level, permits highly informed inferences about the functions on which imprinted gene expression converges. Our findings indicate the neuronal regulation of motivated behaviours such as feeding and sleep, alongside the regulation of pituitary function, as functional hotspots for imprinting. This adds statistical rigour to prior assumptions and provides testable predictions for novel neural and behavioural phenotypes associated with specific genes and imprinted gene networks. In turn, this work sheds further light on the potential evolutionary drivers of genomic imprinting in the brain.

Microglia - mediated immunity partly contributes to the genetic association between Alzheimer's disease and hippocampal volume.

Genome-wide association studies (GWAS) suggest that Alzheimer's disease (AD) is partly explained by a burden of risk alleles (single nucleotide polymorphisms; SNPs) with relatively small effects. However, the mechanisms by which these loci cumulatively confer susceptibility remain largely unknown. Accumulating evidence suggests an association between increased AD risk allele burden (measured via a polygenic risk profile score; AD-RPS) with reduced hippocampal volume (HV) across a number of independent cohorts. These lines of research suggest that the reduced HV may be a causal mechanism of risk in the development of late-onset Alzheimer's disease (AD). However, as RPS assesses broad, cumulative genetic risk, little is known about the biological processes which may explain this observation. Here, we leverage GWAS data from i) 17,008 late onset AD cases & 37,154 controls and ii) hippocampal volume (N = 12,147; N = 9707) to explore putative pathways that may explain this association. We first demonstrate an association between whole genome AD-RPS and HV (PT < 0.5, Z = -2.07, P = 0.038), confirming previous associations. Second, we restrict our analysis to SNPs within AD genes within a microglia mediated immunity network (NGENES = 56). A microglia AD-RPS was further associated with HV (PT < 0.01; Z = -2.152, P = 0.031). Last, using a competitive, permutation based approach, we show that the common variation within this candidate gene-set is associated with HV, controlling for SNP set-size (P = 0.024). Together, the observations suggest that the relationship between AD and HV is partially explained by genes within an AD-linked microglia mediated immunity network.

Macroinvertebrate Taxonomic and Functional Trait Compositions within Lotic Habitats Affected By River Restoration Practices.

The widespread degradation of lotic ecosystems has prompted extensive river restoration efforts globally, but many studies have reported modest ecological responses to rehabilitation practices. The functional properties of biotic communities are rarely examined within post-project appraisals, which would provide more ecological information underpinning ecosystem responses to restoration practices and potentially pinpoint project limitations. This study examines macroinvertebrate community responses to three projects which aimed to physically restore channel morphologies. Taxonomic and functional trait compositions supported by widely occurring lotic habitats (biotopes) were examined across paired restored and non-restored (control) reaches. The multivariate location (average community composition) of taxonomic and functional trait compositions differed marginally between control and restored reaches. However, changes in the amount of multivariate dispersion were more robust and indicated greater ecological heterogeneity within restored reaches, particularly when considering functional trait compositions. Organic biotopes (macrophyte stands and macroalgae) occurred widely across all study sites and supported a high alpha (within-habitat) taxonomic diversity compared to mineralogical biotopes (sand and gravel patches), which were characteristic of restored reaches. However, mineralogical biotopes possessed a higher beta (between-habitat) functional diversity, although this was less pronounced for taxonomic compositions. This study demonstrates that examining the functional and structural properties of taxa across distinct biotopes can provide a greater understanding of biotic responses to river restoration works. Such information could be used to better understand the ecological implications of rehabilitation practices and guide more effective management strategies.

When is the best time to sample aquatic macroinvertebrates in ponds for biodiversity assessment?

Ponds are sites of high biodiversity and conservation value, yet there is little or no statutory monitoring of them across most of Europe. There are clear and standardised protocols for sampling aquatic macroinvertebrate communities in ponds, but the most suitable time(s) to undertake the survey(s) remains poorly specified. This paper examined the aquatic macroinvertebrate communities from 95 ponds within different land use types over three seasons (spring, summer and autumn) to determine the most appropriate time to undertake sampling to characterise biodiversity. The combined samples from all three seasons provided the most comprehensive record of the aquatic macroinvertebrate taxa recorded within ponds (alpha and gamma diversity). Samples collected during the autumn survey yielded significantly greater macroinvertebrate richness (76% of the total diversity) than either spring or summer surveys. Macroinvertebrate diversity was greatest during autumn in meadow and agricultural ponds, but taxon richness among forest and urban ponds did not differ significantly temporally. The autumn survey provided the highest measures of richness for Coleoptera, Hemiptera and Odonata. However, richness of the aquatic insect order Trichoptera was highest in spring and lowest in autumn. The results illustrate that multiple surveys, covering more than one season, provide the most comprehensive representation of macroinvertebrate biodiversity. When sampling can only be undertaken on one occasion, the most appropriate time to undertake surveys to characterise the macroinvertebrate community biodiversity is during autumn, although this may need to be modified if other floral and faunal groups need to be incorporated into the sampling programme.

Long-term river invertebrate community responses to groundwater and surface water management operations.

River flow regimes have been transformed by groundwater and surface water management operations globally, prompting widespread ecological responses. Yet, empirical evidence quantifying the simultaneous effects of groundwater and surface water management operations on freshwater ecosystems remains limited. This study combines a multi-decadal freshwater invertebrate dataset (1995-2016) with groundwater model outputs simulating the effects of different anthropogenic flow alterations (e.g. groundwater abstraction, effluent water returns) and river discharges. A suite of flow alteration- and flow-ecology relationships were modelled that tested different invertebrate community responses (taxonomic, functional, flow response guilds, individual taxa). Most flow alteration-ecology relationships were not statistically significant, highlighting the absence of consistent, detectable ecological responses to long-term water management operations. A small number of significant statistical models provided insights into how flow alterations transformed specific ecological assets; including Ephemeroptera, Plecoptera and Trichoptera taxa which are rheophilic in nature being positively associated with groundwater abstraction effects reducing river discharges by 0-15%. This represents a key finding from a water resource management operation perspective given that such flow alteration conditions were observed on average in over two-thirds of the study sites examined. In a small number of instances, specific invertebrate responses displayed relative declines associated with the most severe groundwater abstraction effects and artificial hydrological inputs (predominantly effluent water returns). The strongest flow-ecology relationships were recorded during spring months, when invertebrate communities were most responsive to antecedent minimum and maximum discharges, and average flow conditions in the preceding summer months. Results from this study provide new evidence indicating how groundwater and surface water resources can be managed to conserve riverine ecological assets. Moreover, the ensemble of flow alteration- and flow-ecology relationships established in this study could be used to guide environmental flow strategies. Such findings are of global importance given that future climatic change and rising societal water demands are likely to further transform river flow regimes and threaten freshwater ecosystems.

Influence of diets high and low in animal fat on bowel habit, gastrointestinal transit time, fecal microflora, bile acid, and fat excretion.

Epidemiological observations and animal experiments suggest that large bowel cancer is related to serveral factors. Among them, high dietary intakes of animal fat, the presence in the colon of relatively high levels of bile acids, specific patterns of intestinal microflora, slow transit through the gut, and low stool weights. Under metabolic conditions we have observed the effect on these variables of dietes containing 62 or 152 g/day of fat mainly of animal origin in six healthy young men over 4-wk periods. No change attributable to the diet was observed in the subjects' bowel habit, fecal weight, mean transit time through the gut, or in the excretion of dry matter. Total fecal bile acid excretion was significantly higher on the high fat diet (320 +/- 120 mg/day) than on the low fat diet (139.7) +/- 63 mg/day) t test = 7.78 P less than 0.001 as also was the total fecal fatty acid excretion, 3.1+/-0.71 and 1.14+/-0.35 g/day, respectively t test = 11.4 P less than 0.001). The fecal microflora including the nuclear dehydrogenating clostridia were unaltered by the dietary changes as was fecal beta-glucuronidase activity. Dietary changes which increase animal fat intake clearly influence fecal bile acid excretion in a way that would favor the development of large bowel cancer if current theories prove to be true. Dietary fat however has no effect on overall colonic function so other components of the diet must be responsible for the observed associations of bowel cancer with slow transit and reduced fecal bulk.

MiR-137-derived polygenic risk: effects on cognitive performance in patients with schizophrenia and controls.

Variants at microRNA-137 (MIR137), one of the most strongly associated schizophrenia risk loci identified to date, have been associated with poorer cognitive performance. As microRNA-137 is known to regulate the expression of ~1900 other genes, including several that are independently associated with schizophrenia, we tested whether this gene set was also associated with variation in cognitive performance. Our analysis was based on an empirically derived list of genes whose expression was altered by manipulation of MIR137 expression. This list was cross-referenced with genome-wide schizophrenia association data to construct individual polygenic scores. We then tested, in a sample of 808 patients and 192 controls, whether these risk scores were associated with altered performance on cognitive functions known to be affected in schizophrenia. A subgroup of healthy participants also underwent functional imaging during memory (n=108) and face processing tasks (n=83). Increased polygenic risk within the empirically derived miR-137 regulated gene score was associated with significantly lower performance on intelligence quotient, working memory and episodic memory. These effects were observed most clearly at a polygenic threshold of P=0.05, although significant results were observed at all three thresholds analyzed. This association was found independently for the gene set as a whole, excluding the schizophrenia-associated MIR137 SNP itself. Analysis of the spatial working memory fMRI task further suggested that increased risk score (thresholded at P=10-5) was significantly associated with increased activation of the right inferior occipital gyrus. In conclusion, these data are consistent with emerging evidence that MIR137 associated risk for schizophrenia may relate to its broader downstream genetic effects.

Diet and the human intestinal bacterial flora.

In this review, the factors thought to be important in determining the composition of the gut bacterial flora are discussed. In the light of these, the effects of various dietary manipulations on the composition and metabolic activity of the gut flora are described. In general, the main effects of diet would be expected to be manifested in the right colon, whereas the material available for investigation is feces. Consequently, the data that are available tend to underestimate the effects of diet.

Metabolic epidemiology of dietary factors in large bowel cancer.

According to the hypothesis being tested by this laboratory, bacteria (in particular, certain clostridia) metabolize the bile acids to give unsaturated products that are important in the causation of colorectal cancer. In this paper, various dietary regimens are discussed in terms of their effect on the fecal steroid concentration and on the gut bacterial flora. The diets considered include high- and low-fiber, high- and low-meat, and high- and low-fat diets.

The distribution of small and intermediate conductance calcium-activated potassium channels in the rat sensory nervous system.

Small (SK) and intermediate (IK) conductance calcium-activated potassium channels are candidate ion channels for the regulation of excitability in nociceptive neurones. We have used unique peptide-directed antisera to describe the immunocytochemical distribution of the known isoforms of these ion channels in dorsal root ganglia (DRG) and spinal cord of the rat. These investigations sought to characterize further the phenotype and hence possible functions of nociceptive neurone subpopulations in the rat. In addition, using Western blotting, we sought to determine the level of protein expression of SK and IK channels in sensory nervous tissues following induction of inflammation (Freund's Complete Adjuvant (FCA) arthritis model) or nerve injury (chronic constriction injury model). We show that SK1, SK2, SK3 and IK1 are all expressed in DRG and spinal cord. Morphometric analysis revealed that SK1, SK2 and IK1 were preferentially localized to neurones having cell bodies <1000 microm2 (putative nociceptors) in DRG. Dual labeling immunocytochemistry showed that these ion channels co-localize with both CGRP and IB4, known markers of nociceptor sub-populations. SK2 was localized almost exclusively in the superficial laminae of the spinal cord dorsal horn, the region in which many sensory afferents terminate; the distribution of SK1 and IK1 was more widespread in spinal cord, although some preferential labeling within the dorsal horn was observed in the case of IK1. Here we show evidence for a distinctive pattern of expression for certain members of the calcium-activated potassium channel family in the rat DRG.

Changes in fecal composition and colonic function due to cereal fiber.

The effect on colonic function of adding wheat fiber for 3 weeks to the metabolically-controlled diets of six healthy volunteers has been studied. Increasing dietary fiber intake from 17 to 45 g/day increased fecal weight from 79 +/- 6.6 g/day to 228 +/- 29.9 g/day and shortened mean transit time, measured by a continuous marker method, from 57.8 +/- 8.3 hr to 40.3 +/- 8.9 hr. The increase in fecal weight was largely due to water. Fiber caused a dilution of fecal marker and an increase in fecal fat, nitrogen, and calcium output. Fecal sodium, potassium, and chloride showed only small changes but volatile fatty acid output increased significantly without concentrations changing. Fecal bile acid output increased from 199 +/- 46 mg/day to 279 +/- 46 mg/day. These changes are discussed in light of current views of the role of dietary fiber in protecting against colon cancer.

Diet and fecal steroid profile in a South Asian population with a low colon-cancer rate.

South Asian immigrants to England and Wales have low mortality from colon cancer and high mortality from coronary heart disease compared with the general population. In a survey of a predominantly Gujarati population in northwest London, both vegetarians and nonvegetarians had similar total dietary fat intake to the native British population but higher dietary fiber intake. Total fecal bile acid and neutral animal sterol concentrations were lower in South Asians than in a native British comparison group. Sixty-two percent of South Asians excreted detectable quantities of free primary bile acids, which were not present in stools from native British subjects. The ratio of fecal coprostanol to total neutral animal sterols was also lower in South Asians. Low risk of colon cancer in this population may be related to reduced microbial activity in the bowel and low levels of tumor-promoting secondary bile acids.

Molecular and clinical risk markers in colon cancer trials.

Although there is a great deal of information concerning the risk factors for colorectal cancer, progress has been slow in determining methods of chemoprevention. In part this is because colorectal cancer consists of a series of events over 10-15 years or more, which affects only 3-5% of the population. Risk markers for use in patient selection are needed to decrease the sample size needed. In addition, there is a need to identify intermediate endpoints of success to decrease the follow-up time. Clinical markers of risk are a history of colorectal adenomas and of inflammatory bowel disease. Molecular markers include a number of genetic markers and enzyme polymorphisms.

Nitrate and human cancer: a review of the evidence.

Nitrites, derived mainly from ingested nitrates, may react in vivo with secondary nitrogen compounds occurring naturally in certain foods to form N-nitroso compounds, which are potent carcinogens in laboratory animals. This paper reviews the mechanism of their formation and the evidence from epidemiological studies that high nitrate ingestion is involved in the aetiology of human cancer.

Dietary salt, nitrate and stomach cancer mortality in 24 countries. European Cancer Prevention (ECP) and the INTERSALT Cooperative Research Group.

BACKGROUND: High salt and nitrate intake are considered as risk factors for stomach cancer, but little is known about possible interactions. This ecological study examines the respective importance of both factors for stomach cancer mortality at the population level using data obtained under standardized conditions and with biochemical analyses performed in the same laboratories. METHODS: Randomly selected 24-hour urine samples from 39 populations, sampled from 24 countries (N = 5756 people for sodium, 3303 for nitrate) were obtained from the INTERSALT study. Median sodium and nitrate levels were age- and sex-standardized between ages 20-49 years and averaged per country. Ecological correlation-regression analyses were done in relation to national stomach cancer mortality rates. RESULTS: The Pearson correlation of stomach cancer mortality with sodium for the 24 countries was: 0.70 in men and 0.74 in women (both P < 0.001) and with nitrate: 0.63 (P = 0.001) in men and (P < 0.005) in women. In multiple regression of stomach cancer mortality, using sodium and nitrate as independent variables the adjusted R2 was 0.61 in men and 0.54 in women (both P < 0.001). Addition of the interaction term (sodium x nitrate) to the previous model increased the adjusted R2 to 0.77 in men, and to 0.63 in women. The analysis of this model showed that the importance of nitrate as risk factor for stomach cancer mortality increased markedly with higher sodium levels. However, the relationship of stomach cancer mortality with sodium was always stronger than with nitrate. CONCLUSIONS: Salt intake, measured as 24-hour urine sodium excretion, is likely the rate-limiting factor of stomach cancer mortality at the population level.

Twenty-four hour urinary nitrate excretion in 48 populations from 30 countries: an ECP-INTERSALT collaborative study.

BACKGROUND: There is considerable interest in the possible role of nitrate in gastric carcinogenesis, but little information on nitrate intake around the world. This is the first study to give comprehensive standardised data on nitrate excretion as a marker of intake, using 48 worldwide population samples. METHODS: Urinary nitrate excretion has been shown to be a valid measure of nitrate intake in people under 50. This report presents data on 24-hour urinary nitrate excretion from urine collections obtained in the INTERSALT study, based on random samples of men and women aged 20-49 from each of 48 population samples in 30 countries. RESULTS: There was large variation in urinary nitrate excretion both within and between samples; within-sample (individual) distributions tended to be skewed towards higher values. Median values of the samples ranged from 0.42 mmol/day (Labrador, Canada) to 3.52 (Beijing, People's Republic of China) in men and 0.44 mmol/day (Colombia) to 3.44 (Beijing) in women. Overall, median values were higher in men than women by 11% on average (higher in men in 37 of 48 population samples). Comparison by geographical region of median values for men and women combined showed relatively low values in the samples in North America and Northern Europe (range 0.46-0.88 mmol/day), slightly higher values in Western Europe and Africa (0.68-1.11), and intermediate to high values in Southern Europe, Eastern and Central Europe and India (0.86-2.47). The highest median values were found in the Far Eastern samples (up to 3.48). Median values in the Central and South American samples ranged from 0.48 mmol/day (Colombia) to 1.37 (Xingu Indians of Brazil, and Argentina). CONCLUSIONS: For the first time, these data give standardized information on urinary nitrate excretion from different geographical regions of the world, and provide a basis for the further exploration of the role of nitrate in the aetiology of disease in human populations.

Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk.

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.

Nutrition and human cancer.

It is now widely accepted that nutrition is as important as tobacco as a cause of human cancer. However, whereas we can dispense with tobacco with general benefit to health, we cannot do without food. It is, therefore, necessary to determine the pattern of nutrition that is associated with the lowest overall cancer risk. This is made more difficult by the fact that some kinds of cancer are associated with overnutrition (e.g., that of the colon, breast, and prostate) and some with poor nutrition (e.g., esophagus, stomach, and liver). The tools to determine this are epidemiology, animal models, in vitro studies, and dietary intervention studies. These all have their strengths and weaknesses, which will be discussed. It is concluded that the major nutrition factors associated with increased cancer risk are: (a) overweight [as a surrogate for excess energy intake]; (b) low intake of fresh fruit; (c) low intake intake of vegetables; and (d) low intake of whole-grain cereals. There have been many other associations with cancer at specific sites noted in the literature. However, it is the overall risk of cancer that must be minimized.