An in vivo neuroimmune organoid model to study human microglia phenotypes.

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, we developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Our data show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, we demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly.

Cardiovascular Safety of Testosterone-Replacement Therapy.

BACKGROUND: The cardiovascular safety of testosterone-replacement therapy in middle-aged and older men with hypogonadism has not been determined. METHODS: In a multicenter, randomized, double-blind, placebo-controlled, noninferiority trial, we enrolled 5246 men 45 to 80 years of age who had preexisting or a high risk of cardiovascular disease and who reported symptoms of hypogonadism and had two fasting testosterone levels of less than 300 ng per deciliter. Patients were randomly assigned to receive daily transdermal 1.62% testosterone gel (dose adjusted to maintain testosterone levels between 350 and 750 ng per deciliter) or placebo gel. The primary cardiovascular safety end point was the first occurrence of any component of a composite of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke, assessed in a time-to-event analysis. A secondary cardiovascular end point was the first occurrence of any component of the composite of death from cardiovascular causes, nonfatal myocardial infarction, nonfatal stroke, or coronary revascularization, assessed in a time-to-event analysis. Noninferiority required an upper limit of less than 1.5 for the 95% confidence interval of the hazard ratio among patients receiving at least one dose of testosterone or placebo. RESULTS: The mean (±SD) duration of treatment was 21.7±14.1 months, and the mean follow-up was 33.0±12.1 months. A primary cardiovascular end-point event occurred in 182 patients (7.0%) in the testosterone group and in 190 patients (7.3%) in the placebo group (hazard ratio, 0.96; 95% confidence interval, 0.78 to 1.17; P<0.001 for noninferiority). Similar findings were observed in sensitivity analyses in which data on events were censored at various times after discontinuation of testosterone or placebo. The incidence of secondary end-point events or of each of the events of the composite primary cardiovascular end point appeared to be similar in the two groups. A higher incidence of atrial fibrillation, of acute kidney injury, and of pulmonary embolism was observed in the testosterone group. CONCLUSIONS: In men with hypogonadism and preexisting or a high risk of cardiovascular disease, testosterone-replacement therapy was noninferior to placebo with respect to the incidence of major adverse cardiac events. (Funded by AbbVie and others; TRAVERSE ClinicalTrials.gov number, NCT03518034.).

A grassroots antiracist program: The motivation and perceived growth of participants in a community-based, intergroup dialogue program.

In the United States, racial segregation still organizes the social lives of most people. This segregation of social life continues reinforcing attitudes and behaviors that sustain racial injustice in the United States. Given the longstanding structural forces sustaining the segregated status quo, why do certain individuals seek out opportunities for 'intentional integration'? And what happens when they do? This qualitative study interviewed racially diverse participants in a community-developed, sustained, and strategic intergroup dialogue program called Touchy Topics Tuesday (TTT), located in St. Louis, Missouri. Overall, participants (N = 30) described three interwoven motivations for involvement in the program-a catalytic moment, a long-term commitment mindset, and/or the influence of their social network. Of all these, participants' social network was the predominant motivating force for individuals across racial lines. Participants also reported three main categories of outcomes: intellectual growth, emotional growth, and relational growth. Each of these categories encompass both attitudinal and behavioral changes. The article interrogates these major findings in the context of the intergroup dialogue literature and studies of attitude change and psychotherapy.

Effects of long-term testosterone treatment on cardiovascular outcomes in men with hypogonadism: Rationale and design of the TRAVERSE study.

BACKGROUND: Testosterone exerts some effects on the cardiovascular system that could be considered beneficial; some other effects may potentially increase the risk of cardiovascular (CV) events. Neither the long-term efficacy nor safety of testosterone treatment has been studied in an adequately-powered randomized trial. METHODS: The Testosterone Replacement therapy for Assessment of long-term Vascular Events and efficacy ResponSE in hypogonadal men (TRAVERSE) study is a randomized, double-blind, placebo-controlled, parallel group, non-inferiority, multicenter study. Eligible participants are men, 45 to 80 years, with serum testosterone concentration <300 ng/dL and hypogonadal symptoms, who have evidence pre-existing CV disease or increased risk of CV disease. Approximately 6,000 subjects will be randomized to either 1.62% transdermal testosterone gel or a matching placebo gel daily for an anticipated duration of up to 5 years. The primary outcome is CV safety defined by the major adverse CV event composite of nonfatal myocardial infarction, nonfatal stroke, or death due to CV causes. The trial will continue until at least 256 adjudicated major adverse CV event endpoints have occurred to assess whether the 95% (2-sided) upper confidence limit for a hazard ratio of 1.5 can be ruled out. Secondary endpoints include prostate safety defined as the incidence of adjudicated high grade prostate cancer and efficacy in domains of sexual function, bone fractures, depression, anemia, and diabetes. RESULTS: As of July 1, 2021, 5,076 subjects had been randomized. CONCLUSIONS: The TRAVERSE study will determine the CV safety and long-term efficacy of testosterone treatment in middle-aged and older men with hypogonadism with or at increased risk of CV disease.

Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Genomic integrity in the male germ line: evidence in support of the disposable soma hypothesis.

The Big Blue λSelect-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis-platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count (P < 0.001) and motility (P < 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated (P < 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver (P < 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former's sentinel position in safeguarding the stability of the genome.

Epididymal CYP2E1 plays a critical role in acrylamide-induced DNA damage in spermatozoa and paternally mediated embryonic resorptions†.

Acrylamide is a ubiquitous toxicant in human lives, due to its formation in many food products. Acrylamide induces dominant lethal mutations with administration of 25 mg/kg bw/day for 5 days in male mice. Cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) is responsible for this dominant lethality. CYP2E1 is the only enzyme responsible for the conversion of acrylamide to the highly reactive metabolite glycidamide, which forms adducts with DNA. CYP2E1 is present predominantly in the liver, as well as the brain, kidney, intestines, and spleen. Within the male mouse reproductive tract, CYP2E1 localizes to spermatocytes. However, embryo resorptions have been demonstrated to occur only with exposure of the late stages of spermatogenesis and spermatozoa. It was determined that CYP2E1 is additionally expressed within the mouse epididymal epithelium, and this localization is responsible for acrylamide-induced dominant lethality. Further, an equivalent profile of CYP2E1 expression was identified in the human reproductive tract. While spermatozoa of both species were also established to possess CYP2E1, this did not contribute to acrylamide-induced DNA damage. In vitro studies strengthened these findings further, revealing that acrylamide exposure only induces DNA damage in human and mouse spermatozoa following metabolism by the mouse epididymal epithelial cell line (mECap18) to glycidamide. These findings emphasize, for the first time, the vital role of the epididymis in the reproductive toxicity associated with acute acrylamide exposure.

Locating advance care planning facilitators in general practice increases consumer participation.

BACKGROUND: Advance care planning (ACP) can positively affect end-of-life care experiences. However, uptake of ACP completion is low. The aim of this study was to investigate whether co-locating ACP facilitators in general practice increased participation METHODS: Barwon Health commenced promoting its ACP program in 2008. Trained ACP facilitators assisted consumers, which usually occurred in the program's community-based consulting rooms. From 2012 onwards, ACP facilitators were co-located with 18 general practices, where they assisted consumers at the point of care. RESULTS: Referrals to the program increased from 2008-11 (n = 2520) to 2012-15 (n = 6847). Between 2012 and 2015, 48% of referrals to the program were from the 18 general practices with co-located ACP facilitators, and 93% of these referrals resulted in ACPs completed, compared with 74% from practices without co-located facilitators and 55% from all other sources (P DISCUSSION: Co-locating ACP facilitators in general practice increased the number of referrals to the program and produced higher plan completion rates.

"Time with Babe": Seeing Fetal Remains after Pregnancy Termination for Impairment.

Some North American hospitals now offer parents the opportunity to see, hold, and photograph fetal remains after pregnancy loss. I explore the social, material, and interpretive strategies mobilized to create this fetal visibility after second trimester-induced abortion for fetal anomaly. My analysis examines both the discursive framing of fetal remains in practice guidelines on pregnancy loss and the responses of a group of Canadian women to being offered "time with babe." I show that while guidelines tend to frame contact with fetal remains as a response to women's desires to see their baby and to feel like mothers, women's experiences of this contact were shaped by more diverse wishes and concerns as well as by specific abortion practices and practitioner comments and actions.

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.

Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation.

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

Electrophilic aldehydes generated by sperm metabolism activate mitochondrial reactive oxygen species generation and apoptosis by targeting succinate dehydrogenase.

Oxidative stress is a major cause of defective sperm function in cases of male infertility. Such stress is known to be associated with high levels of superoxide production by the sperm mitochondria; however, the causes of this aberrant activity are unknown. Here we show that electrophilic aldehydes such as 4-hydroxynonenal (4HNE) and acrolein, generated as a result of lipid peroxidation, target the mitochondria of human spermatozoa and stimulate mitochondrial superoxide generation in a dose- and time-dependent manner. The activation of mitochondrial electron leakage by 4HNE is shown to involve the disruption of succinate dehydrogenase activity and subsequent activation of an intrinsic apoptotic cascade beginning with a loss of mitochondrial membrane potential and terminating in oxidative DNA adduct formation, DNA strand breakage, and cell death. A tight correlation between spontaneous mitochondrial superoxide generation and 4HNE content (R(2) = 0.89) in untreated populations of human spermatozoa emphasized the pathophysiological significance of these findings. The latter also provide a biochemical explanation for the self-perpetuating nature of oxidative stress in the male germ line, with the products of lipid peroxidation stimulating free radical generation by the sperm mitochondria in a positive feedback loop.

Risk Stratification to Support Antibiotic Stewardship and Breastfeeding Exclusivity in a Military Treatment Facility.

OBJECTIVE: To safely reduce unnecessary antibiotic exposure in neonates exposed to chorioamnionitis and inadequately treated Group B Streptococcus (GBS) using the early-onset sepsis (EOS) calculator for risk stratification and a 36-hour antibiotic duration. DESIGN: Evidence-based quality improvement initiative. SETTING/LOCAL PROBLEM: Obstetric service at a midsized military treatment facility with approximately 2,000 births annually and no standard process for neonatal EOS risk assessment. PARTICIPANTS: Clinical nurse specialist, physicians, nursing leadership, unit-level nursing champions, and nurses assigned to the mother-baby and labor and delivery units. INTERVENTION/MEASUREMENTS: An interdisciplinary working group created a protocol to institute an EOS risk assessment calculator, a note for the electronic heath record, and interdisciplinary education for all staff providing care to neonates in our facility. RESULTS: Before implementation of the EOS calculator, 97.6% of neonates exposed to chorioamnionitis or inadequate maternal GBS treatment received antibiotics; after implementation, the mean rate dropped to 32%. Exclusive breastfeeding rates before discharge in neonates exposed to chorioamnionitis or inadequate maternal GBS treatment also increased during this time, from 40% to a mean of 89%. After implementation, there were no readmissions to our institution for culture-proven sepsis within 14 days of discharge. CONCLUSION: Multidisciplinary team-led implementation of the EOS calculator and of shortened antibiotic duration were associated with safely reduced antibiotic exposure in well-appearing neonates exposed to chorioamnionitis and GBS. In addition, dramatically improved rates of exclusive breastfeeding at discharge were observed in this population.

Attitudes to ageing amongst health care professionals: a qualitative systematic review.

OBJECTIVES: Older adults utilising healthcare may be vulnerable to systemic bias regarding ageing potentially affecting care. Primary aim is to systematically review the qualitative literature examining attitudes to ageing amongst health care professionals (HCPs). Secondary aim is to describe and compare attitudes to ageing between different professional groups. METHODS: Studies examining attitudes to ageing of HCPs were identified using four databases. Original qualitative research with HCPs was included, excluding studies examining students alone, reviews, and grey literature. Three authors undertook standardised data extraction and quality rating for each study and thematic synthesis, with a fourth senior author reviewing. Quality appraisal followed the Attree and Milton guidelines (2006), including studies rated A-C. RESULTS: Of 5869 citations, 13 met initial inclusion criteria. Synthesis of the 12 studies graded A-C followed (excluding one study rated-D). Nurses, doctors, and social workers were most frequently investigated. Identified themes included Attitudes towards older persons, The role of the older persons' family, Behaviour of HCPs towards older persons, Behaviour of older persons towards HCPs, and Definitions of an older person. An overarching theme emerged of the systemic context of attitudes to ageing. Common attitudes were found across disciplines, with doctors emphasising complexity and dependency and nurses the burden of care. CONCLUSIONS: HCP's attitudes to ageing vary and are shaped by professional experiences and systemic factors. HCP attitudes may affect responses to and care of the older person. Therefore, interprofessional research, and systemic and service changes across disciplines are required to foster real and sustainable attitudinal change to support older peoples' care.

Basal body organization and cell geometry during the cell cycle in Tetrahymena thermophila.

Tetrahymena thermophila possesses arrays of motile cilia that promote fluid flow for cell motility. These consist of intricately organized basal bodies (BBs) that nucleate and position cilia at the cell cortex. Tetrahymena cell geometry and spatial organization of BBs play important roles in cell size, swimming, feeding, and division. How cell geometry and BB organization are established and maintained remains poorly understood, and prior studies have been limited due to difficulties in accurate BB identification and small sample size. We therefore developed an automated image processing pipeline that segments single cells, distinguishes unique BB populations, assigns BBs into distinct ciliary rows, and distinguishes new from mature BBs. We identified unique features to describe the variation of cell shape and BB spatial organization in unsynchronized single-cell images. The results reveal asymmetries in BB distribution and ingression of the cytokinetic furrow within the cell. Moreover, we establish novel spatial and temporal waves in new BB assembly through the cell cycle. Finally, we used measurements from single cells across the cell cycle to construct a generative model that allows synthesis of movies depicting single cells progressing through the cell cycle. Our approach is expected to be of particular value for characterizing Tetrahymena mutants.

Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols.

The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa.

Human spermatozoa are characterized by poor functionality and abundant DNA damage that collude to generate the high incidences of male infertility and miscarriage seen in our species. Although apoptosis has been suggested as a possible cause of poor sperm quality, the ability of these cells to enter an apoptotic state and the factors that might trigger such an event are unresolved. In the present study we provide evidence that the commitment of these cells to apoptosis is negatively regulated by PI3K (phosphoinositide 3-kinase)/AKT. If PI3K activity is inhibited, then spermatozoa default to an apoptotic cascade characterized by rapid motility loss, mitochondrial reactive oxygen species generation, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolization and oxidative DNA damage. However, the specialized physical architecture of spermatozoa subsequently prevents endonucleases activated during this process from penetrating the sperm nucleus and cleaving the DNA. As a result, DNA fragmentation does not occur as a direct result of apoptosis in spermatozoa as it does in somatic cells, even though oxidative DNA adducts can clearly be detected. We propose that this unusual truncated apoptotic cascade prepares spermatozoa for silent phagocytosis within the female tract and prevents DNA-damaged spermatozoa from participating in fertilization.

Apnea of prematurity and sudden infant death syndrome.

Apnea is a frequent occurrence in prematurity and its prevalence in the most severely preterm population is indicative of an immature respiratory neural control system. Preterm infants are also at increased risk of Sudden Infant Death Syndrome (SIDS), which has been associated with similar respiratory neural control dysfunction seen in prematurity. Generally, abnormalities in both central and peripheral mechanisms of respiratory control are thought to be key underlying features of abnormal respiratory system development. Numerous factors contribute to the etiology of apnea and respiratory control dysfunction including the environment (e.g., substance use/misuse), sex, genetics, a vulnerable neonate, and various underlying comorbidities. However, there are major gaps in our understanding of both normal and abnormal respiratory control system development, which highlights the need for continued research using novel and innovative methods.

Comparison of psychedelic and near-death or other non-ordinary experiences in changing attitudes about death and dying.

Both psychedelic drug experiences and near-death experiences can occasion changes in perspectives on death and dying, but there have been few direct comparisons of these phenomena. This study directly compared psychedelic occasioned and non-drug experiences which altered individuals' beliefs about death. Individuals who reported an experience that altered their beliefs about death occasioned by either a psychedelic drug or a near-death or other non-ordinary experience completed an online survey. Circumstances of the experience, mystical and near-death subjective features, changes in attitudes about death, and other persisting effects were evaluated. The study sample (n = 3192) included five groups: non-drug near-death or other non-ordinary experiences (n = 933), and drug experiences occasioned by lysergic acid diethylamide (LSD) (n = 904), psilocybin (n = 766), ayahuasca (n = 282), or N,N-dimethyltryptamine (DMT) (n = 307). Analyses of differences in experiences were adjusted statistically for demographic differences between groups. Compared to the psychedelic groups, the non-drug group was more likely to report being unconscious, clinically dead, and that their life was in imminent danger. The groups were remarkably similar in the reported changes in death attitudes attributed to the experience, including a reduced fear of death and high ratings of positive persisting effects and personal meaning, spiritual significance, and psychological insight. Although both psychedelic and non-drug participants showed robust increases on standardized measures of mystical and near-death experiences, these measures were significantly greater in the psychedelic participants. Non-drug participants were more likely to rate their experiences as the single most meaningful of their lives. Comparing across psychedelic substances, ayahuasca and DMT groups tended report stronger and more positive enduring consequences of the experience than the psilocybin and LSD groups, which were largely indistinguishable. These data provide a detailed characterization and comparison of psychedelic occasioned and non-drug experiences that changed attitudes about death and suggest the importance of future prospective psychedelic administration studies.