A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells.

In normal tissue homeostasis, bidirectional communication between different cell types can shape numerous biological outcomes. Many studies have documented instances of reciprocal communication between fibroblasts and cancer cells that functionally change cancer cell behavior. However, less is known about how these heterotypic interactions shape epithelial cell function in the absence of oncogenic transformation. Furthermore, fibroblasts are prone to undergo senescence, which is typified by an irreversible cell cycle arrest. Senescent fibroblasts are also known to secrete various cytokines into the extracellular space; a phenomenon that is termed the senescence-associated secretory phenotype (SASP). While the role of fibroblast-derived SASP factors on cancer cells has been well studied, the impact of these factors on normal epithelial cells remains poorly understood. We discovered that treatment of normal mammary epithelial cells with conditioned media from senescent fibroblasts (SASP CM) results in a caspase-dependent cell death. This capacity of SASP CM to cause cell death is maintained across multiple senescence-inducing stimuli. However, the activation of oncogenic signaling in mammary epithelial cells mitigates the ability of SASP CM to induce cell death. Despite the reliance of this cell death on caspase activation, we discovered that SASP CM does not cause cell death by the extrinsic or intrinsic apoptotic pathway. Instead, these cells die by an NLRP3, caspase-1, and gasdermin D-dependent induction of pyroptosis. Taken together, our findings reveal that senescent fibroblasts can cause pyroptosis in neighboring mammary epithelial cells, which has implications for therapeutic strategies that perturb the behavior of senescent cells.

Alcohol use disorder, cannabis use disorder, and eating disorder symptoms among male and female college students.

BACKGROUND AND OBJECTIVES: Eating disorders (EDs) and substance use disorders are prevalent among college students in the United States, with underlying common mechanisms suggesting co-occurrence of these in the student population. As treatment prognosis of EDs improves when they are identified and treated with early intervention, it is essential to understand which substance use behaviors associate with EDs in students. METHODS: Using a sample of 471 college students recruited for a study on high risk drinking (i.e., students needed to pregame regularly to be included), we explored the associations between ED symptomatology and two common substances used in this population: alcohol and cannabis. As most research on EDs focuses on female students only or does not separate out males and females, we examined whether sex assigned at birth moderated the association between ED symptomatology and substance use outcomes. RESULTS: About one-third (32.4%) of the sample screened positive for an ED, with females significantly more likely to screen positive. Males were significantly more likely to screen positive for an alcohol or cannabis use disorder. Screening positive for an ED associated with cannabis use frequency and cannabis use disorder symptoms, but not with alcohol outcomes. Sex moderated the association between ED and cannabis use disorder symptoms, with positive ED screen male students experiencing the highest cannabis use disorder symptoms. DISCUSSION AND CONCLUSIONS: It is necessary to further assess how sex differences in substance use and ED symptomatology inform each other. SCIENTIFIC SIGNIFICANCE: Findings underscore the need to assess and screen for cannabis use disorder among students who screen positive for an ED, and, more specifically, with focused attention on male students with ED symptoms.

Evaluating changes to home bowel cancer screening kits: an end-user perspective study.

PURPOSE: Many people do not participate in mail-out bowel cancer screening programs due to difficulties using the screening kit. The current study investigated the ways the screening kit could be modified to improve usability. METHODS: 1,109 people evaluated 15 different screening kit modifications. Participants reported on how these kit modifications would affect their screening barriers, their future screening intentions, and how much they would recommend that the modification is made to the current screening kit used the program. All responses were given via an online survey conducted between April and December of 2021. RESULTS: Seventeen percent of previous NBCSP non-participators indicated that a one-sample test would increase their intention to participate. Recommendation ratings demonstrated higher levels of support for modifications that included providing a barcode naming label (M = 9.06, 95% CI [8.81, 9.31]), having a larger diameter opening of the collection tube (M = 8.42, 95% CI [8.10, 8.74]), and highlighting the expiry date on the kit packaging (M = 8.59, 95% CI [8.29, 8.89]). There were lower levels of support for modifications that reduced the size of the packaging the kit is sent in (M = 6.47, 95% CI [6.09, 6.85]), removed branding from kit packaging (M = 5.98, 95% CI [5.57, 6.39]), and removed the information booklet that comes with the screening kit (M = 5.25, 95% CI [4.78, 5.72]). CONCLUSION: These findings highlight multiple ways in which bowel cancer screening kits can be changed to increase usability for invitees of national bowel cancer screening programs. Findings have implications for all screening programs that use immunochemical-based bowel cancer screening kits.

The expanding fireball of Nova Delphini 2013.

A classical nova occurs when material accreting onto the surface of a white dwarf in a close binary system ignites in a thermonuclear runaway. Complex structures observed in the ejecta at late stages could result from interactions with the companion during the common-envelope phase. Alternatively, the explosion could be intrinsically bipolar, resulting from a localized ignition on the surface of the white dwarf or as a consequence of rotational distortion. Studying the structure of novae during the earliest phases is challenging because of the high spatial resolution needed to measure their small sizes. Here we report near-infrared interferometric measurements of the angular size of Nova Delphini 2013, starting one day after the explosion and continuing with extensive time coverage during the first 43 days. Changes in the apparent expansion rate can be explained by an explosion model consisting of an optically thick core surrounded by a diffuse envelope. The optical depth of the ejected material changes as it expands. We detect an ellipticity in the light distribution, suggesting a prolate or bipolar structure that develops as early as the second day. Combining the angular expansion rate with radial velocity measurements, we derive a geometric distance to the nova of 4.54 ± 0.59 kiloparsecs from the Sun.

Modal noise in an integrated photonic lantern fed diffraction-limited spectrograph.

In an attempt to develop a streamlined astrophotonic instrument, we demonstrate the realization of an all-photonic device capable of both multimode to single mode conversion and spectral dispersion on an 8-m class telescope with efficient coupling. The device was a monolithic photonic spectrograph which combined an integrated photonic lantern and an efficient arrayed waveguide grating device. During on-sky testing, we discovered a previously unreported type of noise that made spectral extraction and calibration extremely difficult. The source of the noise was traced to a wavelength-dependent loss mechanism between the feed fiber's multimode near-field pattern and the modal acceptance profile of the integrated photonic lantern. Extensive modeling of the photonic components replicates the wavelength-dependent loss, and demonstrates an identical effect on the final spectral output. We outline that this could be mitigated by directly injecting into the integrated photonic lantern.

Reducing Alcohol Misuse and Promoting Treatment Initiation Among Veterans Through a Brief Internet-Based Intervention: Protocol for a Randomized Controlled Trial.

BACKGROUND: Young adult veterans who served after the September 11 attacks on the United States in 2001 (ie, post-9/11) are at heightened risk for experiencing behavioral health distress and disorders including hazardous drinking, posttraumatic stress disorder, and depression. These veterans often face significant barriers to behavioral health treatment, and reaching them through brief mobile phone-based interventions may help reduce drinking and promote treatment engagement. OBJECTIVE: Following a successful pilot study, this randomized controlled trial (RCT) aims to further test the efficacy of a brief (ie, single session) mobile phone-delivered personalized normative feedback intervention enhanced with content to promote treatment engagement. METHODS: We will conduct an RCT with 800 post-9/11 young adult veterans (aged 18 to 40 years) with potentially hazardous drinking and who have not recently received treatment for any behavioral health problems. Participants will be randomly assigned to the personalized intervention or a control condition with resources for seeking care. The personalized normative feedback module in the intervention focuses on the correction of misperceived norms of peer alcohol use and uses empirically informed approaches to increase motivation to address alcohol use and co-occurring behavioral health problems. Past 30-day drinking, alcohol-related consequences, and treatment-seeking behaviors will be assessed at baseline and 3, 6, 9, and 12 months post intervention. Sex, barriers to care, posttraumatic stress disorder, depression, and severity of alcohol use disorder symptoms will be explored as potential moderators of outcomes. RESULTS: We expect recruitment to be completed within 6 months, with data collection taking 12 months for each enrolled participant. Analyses will begin within 3 months of the final data collection point (ie, 12 months follow-up). CONCLUSIONS: This RCT will evaluate the efficacy of a novel intervention for non-treatment-seeking veterans who struggle with hazardous drinking and possible co-occurring behavioral health problems. This intervention has the potential to improve veteran health outcomes and overcome significant barriers to treatment. TRIAL REGISTRATION: ClinicalTrials.gov NCT04244461; https://clinicaltrials.gov/study/NCT04244461. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/59993.

High fat diet-induced maternal obesity in mice impairs peripartum maternal behaviour.

Obesity during pregnancy represents a significant health issue and can lead to increased complications during pregnancy and impairments with breastfeeding, along with long-term negative health consequences for both mother and offspring. In rodent models, diet-induced obesity (DIO) during pregnancy leads to poor outcomes for offspring. Using a DIO mouse model, consisting of feeding mice a high fat diet for 8 weeks before mating, we recapitulate the effect of high pup mortality within the first 3 days postpartum. To examine the activity of the dam around the time of birth, late pregnant control and DIO dams were recorded in their home cages and the behaviour of the dam immediately before and after birth was analysed. Prior to giving birth, DIO dams spent less time engaging in nesting behaviour, while after birth, DIO dams spent less time in the nest with their pups compared to control dams, indicating reduced pup-engagement in the early postpartum period. We have previously reported that lactogenic hormone action, mediated by the prolactin receptor, in the medial preoptic area of the hypothalamus (MPOA) is critical for the onset of normal postpartum maternal behaviour. We hypothesized that DIO dams may have lower lactogenic hormone activity during late pregnancy, which would contribute to impaired onset of normal postpartum maternal behaviour. Day 16 lactogenic activity, transport of prolactin into the brain, and plasma prolactin concentrations around birth were all similar in control and DIO dams. Moreover, endogenous pSTAT5, a marker of prolactin receptor activity, in the MPOA was unaffected by DIO. Overall, these data indicate that lactogenic activity in late pregnancy of DIO dams is not different to controls and is unlikely to play a major role in impaired onset of normal postpartum maternal behaviour.

A role for fibroblast-derived SASP factors in the activation of pyroptotic cell death in mammary epithelial cells.

In normal tissue homeostasis, bidirectional communication between different cell types can shape numerous biological outcomes. Many studies have documented instances of reciprocal communication between fibroblasts and cancer cells that functionally change cancer cell behavior. However, less is known about how these heterotypic interactions shape epithelial cell function in the absence of oncogenic transformation. Furthermore, fibroblasts are prone to undergo senescence, which is typified by an irreversible cell cycle arrest. Senescent fibroblasts are also known to secrete various cytokines into the extracellular space; a phenomenon that is termed the senescence-associated secretory phenotype (SASP). While the role of fibroblast derived SASP factors on cancer cells has been well studied, the impact of these factors on normal epithelial cells remains poorly understood. We discovered that treatment of normal mammary epithelial cells with conditioned media (CM) from senescent fibroblasts (SASP CM) results in a caspase-dependent cell death. This capacity of SASP CM to cause cell death is maintained across multiple senescence-inducing stimuli. However, the activation of oncogenic signaling in mammary epithelial cells mitigates the ability of SASP CM to induce cell death. Despite the reliance of this cell death on caspase activation, we discovered that SASP CM does not cause cell death by the extrinsic or intrinsic apoptotic pathway. Instead, these cells die by an NLRP3, caspase-1, and gasdermin D (GSDMD)-dependent induction of pyroptosis. Taken together, our findings reveal that senescent fibroblasts can cause pyroptosis in neighboring mammary epithelial cells, which has implications for therapeutic strategies that perturb the behavior of senescent cells.

Oncogenic signaling inhibits c-FLIPL expression and its non-apoptotic function during ECM-detachment.

Inhibition of programmed cell death pathways is frequently observed in cancer cells where it functions to facilitate tumor progression. However, some proteins involved in the regulation of cell death function dichotomously to both promote and inhibit cell death depending on the cellular context. As such, understanding how cell death proteins are regulated in a context-dependent fashion in cancer cells is of utmost importance. We have uncovered evidence that cellular FLICE-like Inhibitory Protein (c-FLIP), a well-known anti-apoptotic protein, is often downregulated in tumor tissue when compared to adjacent normal tissue. These data argue that c-FLIP may have activity distinct from its canonical role in antagonizing cell death. Interestingly, we have discovered that detachment from extracellular matrix (ECM) serves as a signal to elevate c-FLIP transcription and that oncogenic signaling blocks ECM-detachment-induced c-FLIP elevation. In addition, our data reveal that downregulation of c-FLIP promotes luminal filling in mammary acini and that c-FLIP overexpression in cancer cells inhibits colony formation in cells exposed to ECM-detachment. Taken together, our study reveals an unexpected, non-apoptotic role for c-FLIP during ECM-detachment and raises the possibility that c-FLIP may have context-dependent roles during tumorigenesis.

Calmodulin binds to chick lens gap junction protein in a calcium-independent manner.

A biochemically active conjugate of calmodulin and tetramethylrhodamine isothiocyanate (CaM-RITC) was synthesized. When incubated with sections of chick lens, this conjugate bound to the surface membranes of lens fiber cells in the presence of absence of calcium. Incubation of lens sections with antibodies to gap junction protein of lens completely blocked the binding of the conjugate to cell membranes, whereas serum from nonimmunized animals or antibodies to others lens proteins reduced the binding only slightly. By means of a gel overlay procedure, 125I-labeled calmodulin was found to bind to the gap junction protein of lens, also in a calcium-independent manner. These results support the concept that calmodulin may interact with and regulate gap junctions in living cells.

Distinct receptors underlie glutamatergic signalling in inspiratory rhythm-generating networks and motor output pathways in neonatal rat.

Despite the enormous diversity of glutamate (Glu) receptors and advances in understanding recombinant receptors, native Glu receptors underlying functionally identified inputs in active systems are poorly defined in comparison. In the present study we use UBP-302, which antagonizes GluR5 subunit-containing kainate (KA) receptors at < or = 10 microm, but other KA and AMPA receptors at > or = 100 microm, and rhythmically active in vitro preparations of neonatal rat to explore the contribution of non-NMDA receptor signalling in rhythm-generating and motor output compartments of the inspiratory network. At 10 microm, UBP-302 had no effect on inspiratory burst frequency or amplitude. At 100 microm, burst amplitude recorded from XII, C1 and C4 nerve roots was significantly reduced, but frequency was unaffected. The lack of a frequency effect was confirmed when local application of UBP-302 (100 microm) into the pre-Bötzinger complex (preBötC) did not affect frequency but substance P evoked a 2-fold increase. A UBP-302-sensitive (10 microm), ATPA-evoked frequency increase, however, established that preBötC networks are sensitive to GluR5 activation. Whole-cell recordings demonstrated that XII motoneurons also express functional GluR5-containing KA receptors that do not contribute to inspiratory drive, and confirmed the dose dependence of UBP-302 actions on KA and AMPA receptors. Our data provide the first evidence that the non-NMDA (most probably AMPA) receptors mediating glutamatergic transmission within preBötC inspiratory rhythm-generating networks are pharmacologically distinct from those transmitting drive to inspiratory motoneurons. This differential expression may ultimately be exploited pharmacologically to separately counteract depression of central respiratory rhythmogenesis or manipulate the drive to motoneurons controlling airway and pump musculature.

The Interoception Sensory Questionnaire (ISQ): A Scale to Measure Interoceptive Challenges in Adults.

Increasing attention is being paid to how adults on the autism spectrum perceive and interpret the interoceptive sense. This 20-item Interoception Sensory Questionnaire represents a single factor scale that can be interpreted as representing confusion about interoceptive bodily states unless these states are extreme (Alexisomia), and has been designed to discriminate across populations (total sample 511 participants). Findings showed that 74% of adults with autism reported interoceptive confusion. Another finding of the study was that as autistic traits increased, interoceptive confusion increased, with adults with diagnosed autism scoring highest on the construct. Implications for physiological self-regulation as well as physical health outcomes are discussed, as well as recommendations for future research.

Enhancing antimicrobial stewardship by strengthening the veterinary drug regulatory framework.

Antimicrobial resistance is a major and growing public health threat. Recently, Health Canada introduced multiple regulatory changes to strengthen the oversight of antimicrobial drugs for veterinary use. These changes aim specifically at increasing control over importation of veterinary drugs and active pharmaceutical ingredients, mandatory reporting of antimicrobial sales data from manufacturers, importers and compounders and facilitating access to low risk veterinary health products. Additional policy changes under existing authorities are also being made to enhance veterinary supervision of antimicrobial use and to remove production claims for food animals from labels of medically important antimicrobial drugs. These important interlinked initiatives are aimed towards enhancing antimicrobial stewardship in Canada to preserve the effectiveness of existing antimicrobials and to protect the health of Canadians.

Mapping the effects of the selective dopamine D2/D3 receptor agonist quinelorane using pharmacological magnetic resonance imaging.

Dopamine agonists with a high affinity for D2 and D3 receptors have a biphasic effect on rodent locomotion, inducing hypolocomotion at low doses and hyperlocomotion at higher doses. Controversy surrounds the role of the D3 receptor in mediating the hypolocomotor response to low agonist doses. This study examines patterns of neuronal activation induced by varying doses of the D2/D3 receptor agonist quinelorane using blood oxygen level dependent (BOLD) pharmacological magnetic resonance imaging (phMRI), and compares them with corresponding behavioural responses. Quinelorane (3 microg/kg) induced hypolocomotion in rats naive to the testing environment, and in phMRI experiments increased neuronal activity within the anterior olfactory nuclei, nucleus accumbens and islets of Calleja, regions containing a high density of D3 receptors. A 30 microg/kg dose of quinelorane resulted in biphasic locomotor effects, with initial hypolocomotion followed by sustained hyperlocomotion. phMRI indicated that this higher dose increased cerebral activity within limbic and olfactory regions, as did the lower drug dose, but induced additional activation in the caudate-putamen and globus pallidus, areas dense in D2 receptors but containing few D3 receptors. The more restricted pattern of activation at low agonist doses and close temporal relationship between behavioural and BOLD signal responses to quinelorane suggest that those nuclei most dense in D3 receptors play a key role in mediating the hypolocomotor effects of quinelorane. However, the presence of D3 receptors in activated brain regions may be coincidental, and further studies are required to show definitively which class of receptors mediates agonist-induced hypolocomotion. In contrast, the activation of D2 receptors within the striatum appears necessary for quinelorane-induced hyperlocomotion.

The impact of lagging strand replication mutations on the stability of CAG repeat tracts in yeast.

We have examined the stability of long tracts of CAG repeats in yeast mutants defective in enzymes suspected to be involved in lagging strand replication. Alleles of DNA ligase (cdc9-1 and cdc9-2) destabilize CAG tracts in the stable tract orientation, i.e., when CAG serves as the lagging strand template. In this orientation nearly two-thirds of the events recorded in the cdc9-1 mutant were tract expansions. While neither DNA ligase allele significantly increases the frequency of tract-length changes in the unstable orientation, the cdc9-1 mutant produced a significant number of expansions in tracts of this orientation. A mutation in primase (pri2-1) destabilizes tracts in both the stable and the unstable orientations. Mutations in a DNA helicase/deoxyribonuclease (dna2-1) or in two RNase H activities (rnh1Delta and rnh35Delta) do not have a significant effect on CAG repeat tract stability. We interpret our results in terms of the steps of replication that are likely to lead to expansion and to contraction of CAG repeat tracts.

After hours care--a qualitative study of GPs' perceptions of risk of violence and effect on service provision.

BACKGROUND: Workplace violence in general practice has been found to be an important problem in the United Kingdom. No research has been undertaken in this area in Australian urban practice. METHOD: Four focus groups involved 18 urban general practitioners and over 9 hours of taped responses were transcribed. The transcripts were coded and subjected to thematic analysis. RESULTS: General practitioners expressed a wide range of risks relating to the provision of after hours care. This makes them apprehensive about participating in it. Those who had experienced violence, or perceived its risk, had limited their participation in after hours care; sometimes completely. DISCUSSION: Structures may be needed to support provision of after hours general practice services.

Arterial response to venous occlusion in normotensive and hypertensive subjects.

Plethysmographic blood flow records made shortly after venous occlusion of the forearm showed a biphasic response, first vasodilator then vasoconstrictor, in both normotensive and hypertensive subjects. The vasodilator component of this response was significantly lower in hypertensive than in normotensive subjects, whereas the vasoconstrictor component was identical. The decreased vasodilator capacity of the forearm resistance vessels in hypertension may indicate structural adaptation of these vessels, while the unaltered vasoconstrictor response is against any increased myogenic activity in the vascular smooth muscle in hypertensive subjects.

Germ cell stimulation of Sertoli cell protein phosphorylation.

Cultures of Sertoli cells were treated with freshly isolated, intact germ cells to determine if germ cells were capable of influencing Sertoli cell function. By using two-dimensional polyacrylamide gel electrophoresis and autoradiography, germ cells were found to increase several-fold the incorporation of [32P]orthophosphate into two phosphoproteins (which we term germ cell-dependent phosphoproteins 1 and 2 or GC1 and GC2) of Sertoli cells. Increased phosphorylation of GC1 and GC2 was rapid, germ cell dose dependent, and calcium dependent. The increased phosphorylation of GC1 appears to involve calmodulin-dependent protein kinase, while phosphorylation of GC2 appears to involve the activation of calcium/phospholipid-dependent protein kinase (protein kinase C). Medium conditioned by germ cells was capable of eliciting the same response in Sertoli cells. Germ cells had no effect on the phosphorylation of proteins in Chinese hamster ovarian cells, but did result in increased phosphorylation of a protein in TR-ST cells, which migrated similarly to GC1 of Sertoli cells. Neither Chinese hamster ovarian nor TR-ST cells had any effect on Sertoli cell protein phosphorylation. These results indicate that germ cells may be directly involved in the local regulation of Sertoli cell function within the seminiferous epithelium. The results further suggest that the mechanism of germ cell-Sertoli cell interaction involves the mobilization of intracellular calcium, activation of Ca2+/calmodulin-dependent protein kinase, and protein kinase C. We infer from these results that germ cell-Sertoli cell interaction may operate via hydrolysis of Sertoli cell membrane phophatidylinositols.

Two-dimensional gel analysis of Sertoli cell protein phosphorylation: effect of short term exposure to follicle-stimulating hormone.

The response of cultured Sertoli cells to short term FSH stimulation was studied to elucidate early events involved in the hormone response of this cell type. The phosphorylation of proteins by [32P]orthophosphate-labeled cells was examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. FSH stimulation resulted in a variety of changes in the phosphoprotein labeling pattern. Within 5 min, unique phosphoproteins appeared in autoradiograms of treated cells. Increased labeling of vimentin was also noted. After 25 min of FSH stimulation, increases and decreases in apparent labeling intensity were evident in additional phosphoproteins that were constitutively labeled in control cultures. Changes in protein patterns in stained gels were also noted after acute hormone treatment. These observations demonstrate that Sertoli cells respond to hormonal stimulation in a detectable manner within 5 min. Cytoskeletal involvement in initial phases of hormone response is indicated. Mechanisms such as increased protein kinase activity, changes in protein kinase substrate affinity, increased protein turnover, or phosphatase activation may all contribute to the early events involved in Sertoli cell hormone response.

Glypican 3 and glypican 4 are juxtaposed in Xq26.1.

Recently, we have shown that mutations in the X-linked glypican 3 (GPC3) gene cause the Simpson-Golabi-Behmel overgrowth syndrome (SGBS; ). The next centromeric gene detected is another glypican, glypican 4 (GPC4), with its 5' end 120763bp downstream of the 3' terminus of GPC3. One recovered GPC4 cDNA with an open reading frame of 1668nt encodes a putative protein containing three heparan sulfate glycosylation signals and the 14 signature cysteines of the glypican family. This protein is 94.3% identical to mouse GPC4 and 26% identical to human GPC3. In contrast to GPC3, which produces a single transcript of 2.3kb and is stringently restricted in expression to predominantly mesoderm-derived tissues, Northern analyses show that GPC4 produces two transcripts, 3.4 and 4.6kb, which are very widely expressed (though at a much higher level in fetal lung and kidney). Interestingly, of 20 SGBS patients who showed deletions in GPC3, one was also deleted for part of GPC4. Thus, GPC4 is not required for human viability, even in the absence of GPC3. This patient shows a complex phenotype, including the unusual feature of hydrocephalus; but because an uncle with SGBS is less affected, it remains unclear whether the GPC4 deletion itself contributes to the phenotype.