LGBTQ Utilization of a Statewide Tobacco Quitline: Engagement and Quitting Behavior, 2010-2022.

INTRODUCTION: Lesbian, gay, bisexual, transgender, and queer/questioning (LGBTQ) individuals use tobacco at disproportionately high rates but are as likely as straight tobacco users to want to quit and to use quitlines. Little is known about the demographics and geographic distribution of LGBTQ quitline participants, their engagement with services, or their long-term outcomes. AIMS AND METHODS: Californians (N = 333 429) who enrolled in a statewide quitline 2010-2022 were asked about their sexual and gender minority (SGM) status and other baseline characteristics. All were offered telephone counseling. A subset (n = 19 431) was followed up at seven months. Data were analyzed in 2023 by SGM status (LGBTQ vs. straight) and county type (rural vs. urban). RESULTS: Overall, 7.0% of participants were LGBTQ, including 7.4% and 5.4% of urban and rural participants, respectively. LGBTQ participants were younger than straight participants but had similar cigarette consumption. Fewer LGBTQ participants reported a physical health condition (42.1% vs. 48.4%) but more reported a behavioral health condition (71.1% vs. 54.5%; both p's < .001). Among both LGBTQ and straight participants, nearly 9 in 10 chose counseling and both groups completed nearly three sessions on average. The groups had equivalent 30-day abstinence rates (24.5% vs. 23.2%; p = .263). Similar patterns were seen in urban and rural subgroups. CONCLUSIONS: LGBTQ tobacco users engaged with and appeared to benefit from a statewide quitline even though it was not LGBTQ community-based. A quitline with staff trained in LGBTQ cultural competence can help address the high prevalence of tobacco use in the LGBTQ community and reach members wherever they live. IMPLICATIONS: This study describes how participants of a statewide tobacco quitline broke down by sexual orientation and gender. It compares participants both by SGM status and by type of county to provide a more complete picture of quitline participation both in urban areas where LGBTQ community-based cessation programs may exist and in rural areas where they generally do not. To our knowledge, it is the first study to compare LGBTQ and straight participants on their use of quitline services and quitting aids, satisfaction with services received, and rates of attempting quitting and achieving prolonged abstinence from smoking.

Re-engagement of Low-Income Smokers in Quitline Services: Effects of Incentives and Method of Contact.

INTRODUCTION: Financial incentives have been shown to improve recruitment of low-income smokers into tobacco quitline services and to improve cessation outcomes. The present study evaluated their use to re-engage low-income smokers who had already used a quitline. AIMS AND METHODS: Randomly selected Medicaid smokers (N = 5200) who had previously enrolled in a quitline were stratified by time since enrollment (3, 6, 9, or 12 months) and randomly assigned in a 2 × 4 factorial design to receive, by mail or telephone, an invitation to reengage, with an offer of no financial incentive or $10, $20, or $40. The primary outcome measure was re-engagement, defined as use of an additional evidence-based quitline service within 90 days. Data were collected from May 2014 to October 2015 and analyzed in 2022. RESULTS: Of 5200 participants invited to reengage in quitline services, 9.3% did so within 90 days, compared to 6.3% of a randomly selected comparison group (n = 22 614, p < .0001). Letters resulted in greater re-engagement than calls (10.9% vs. 7.8%, respectively, p = .0001). Among letters, there was a dose-response relationship between incentive level and re-engagement rates (p = .003). Re-engagement decreased as time since enrollment increased, from 13.7% at 3 months to 5.7% at 12 months (all p's < 0.0001). CONCLUSIONS: Low-income smokers who previously used quitline services can be motivated to reengage in treatment. Mailed letters and automated calls are effective re-engagement strategies. Financial incentives can increase the effectiveness of re-engagement letters. Inviting Medicaid smokers to re-engage with quitline treatment may help to address socioeconomic health disparities and should be standard practice. IMPLICATIONS: Nicotine addiction is a chronic relapsing disorder, yet most cessation services are designed to help smokers through only one quit attempt. Smoking is increasingly concentrated in populations with physical and psychological co-morbidities, which can make quitting more difficult and impact whether smokers reach out for additional help following relapse. This study examined whether the timing, method, and content of an offer for further assistance influenced re-engagement rates for a vulnerable population of smokers-Medicaid beneficiaries. Relapsing smokers are responsive to re-engagement offers as early as three months, but there is a closing window of opportunity to reach them.

Implementation, Maintenance, and Outcomes of an Electronic Referral to a Tobacco Quitline Across Five Health Systems.

INTRODUCTION: Electronic referral (e-referral) to quitlines helps connect tobacco-using patients to free, evidence-based cessation counseling. Little has been published about the real-world implementation of e-referrals across U.S. health systems, their maintenance over time, and the outcomes of e-referred patients. AIMS AND METHODS: Beginning in 2014, the University of California (UC)-wide project called UC Quits scaled up quitline e-referrals and related modifications to clinical workflows from one to five UC health systems. Implementation strategies were used to increase site readiness. Maintenance was supported through ongoing monitoring and quality improvement programs. Data on e-referred patients (n = 20 709) and quitline callers (n = 197 377) were collected from April 2014 to March 2021. Analyses of referral trends and cessation outcomes were conducted in 2021-2022. RESULTS: Of 20 709 patients referred, the quitline contacted 47.1%, 20.6% completed intake, 15.2% requested counseling, and 10.9% received it. In the 1.5-year implementation phase, 1813 patients were referred. In the 5.5-year maintenance phase, volume was sustained, with 3436 referrals annually on average. Among referred patients completing intake (n = 4264), 46.2% were nonwhite, 58.8% had Medicaid, 58.7% had a chronic disease, and 48.8% had a behavioral health condition. In a sample randomly selected for follow-up, e-referred patients were as likely as general quitline callers to attempt quitting (68.5% vs. 71.4%; p = .23), quit for 30 days (28.3% vs. 26.9%; p = .52), and quit for 6 months (13.6% vs. 13.9%; p = .88). CONCLUSIONS: With a whole-systems approach, quitline e-referrals can be established and sustained across inpatient and outpatient settings with diverse patient populations. Cessation outcomes were similar to those of general quitline callers. IMPLICATIONS: This study supports the broad implementation of tobacco quitline e-referrals in health care. To the best of our knowledge, no other paper has described the implementation of e-referrals across multiple U.S. health systems or how they were sustained over time. Modifying electronic health records systems and clinical workflows to enable and encourage e-referrals, if implemented and maintained appropriately, can be expected to improve patient care, make it easier for clinicians to support patients in quitting, increase the proportion of patients using evidence-based treatment, provide data to assess progress on quality goals, and help meet reporting requirements for tobacco screening and prevention.

Effective fisheries management instrumental in improving fish stock status.

Marine fish stocks are an important part of the world food system and are particularly important for many of the poorest people of the world. Most existing analyses suggest overfishing is increasing, and there is widespread concern that fish stocks are decreasing throughout most of the world. We assembled trends in abundance and harvest rate of stocks that are scientifically assessed, constituting half of the reported global marine fish catch. For these stocks, on average, abundance is increasing and is at proposed target levels. Compared with regions that are intensively managed, regions with less-developed fisheries management have, on average, 3-fold greater harvest rates and half the abundance as assessed stocks. Available evidence suggests that the regions without assessments of abundance have little fisheries management, and stocks are in poor shape. Increased application of area-appropriate fisheries science recommendations and management tools are still needed for sustaining fisheries in places where they are lacking.

Endothelial NMDA receptors mediate activity-dependent brain hemodynamic responses in mice.

Dynamic coupling of blood supply with energy demand is a natural brain property that requires signaling between synapses and endothelial cells. Our previous work showed that cortical arteriole lumen diameter is regulated by N-methyl-d-aspartate receptors (NMDARs) expressed by brain endothelial cells. The purpose of this study was to determine whether endothelial NMDARs (eNMDARs) regulate functional hyperemia in vivo. In response to whisker stimulation, regional cerebral blood flow (rCBF) and hemodynamic responses were assessed in barrel cortex of awake wild-type or eNMDAR loss-of-function mice using two-photon microscopy. Hyperemic enhancement of rCBF and vasodilation throughout the vascular network was observed in wild-type mice. eNMDAR loss of function reduced hyperemic responses in rCBF and plasma flux in individual vessels. Discovery of an endothelial receptor that regulates brain hyperemia provides insight into how neuronal activity couples with endothelial cells.

Three pillars of sustainability in fisheries.

Sustainability of global fisheries is a growing concern. The United Nations has identified three pillars of sustainability: economic development, social development, and environmental protection. The fisheries literature suggests that there are two key trade-offs among these pillars of sustainability. First, poor ecological health of a fishery reduces economic profits for fishers, and second, economic profitability of individual fishers undermines the social objectives of fishing communities. Although recent research has shown that management can reconcile ecological and economic objectives, there are lingering concerns about achieving positive social outcomes. We examined trade-offs among the three pillars of sustainability by analyzing the Fishery Performance Indicators, a unique dataset that scores 121 distinct fishery systems worldwide on 68 metrics categorized by social, economic, or ecological outcomes. For each of the 121 fishery systems, we averaged the outcome measures to create overall scores for economic, ecological, and social performance. We analyzed the scores and found that they were positively associated in the full sample. We divided the data into subsamples that correspond to fisheries management systems with three categories of access-open access, access rights, and harvest rights-and performed a similar analysis. Our results show that economic, social, and ecological objectives are at worst independent and are mutually reinforcing in both types of managed fisheries. The implication is that rights-based management systems should not be rejected on the basis of potentially negative social outcomes; instead, social considerations should be addressed in the design of these systems.

Activation of Cannabinoid Receptors Attenuates Endothelin-1-Induced Mitochondrial Dysfunction in Rat Ventricular Myocytes.

Evidence suggests that the activation of the endocannabinoid system offers cardioprotection. Aberrant energy production by impaired mitochondria purportedly contributes to various aspects of cardiovascular disease. We investigated whether cannabinoid (CB) receptor activation would attenuate mitochondrial dysfunction induced by endothelin-1 (ET1). Acute exposure to ET1 (4 hours) in the presence of palmitate as primary energy substrate induced mitochondrial membrane depolarization and decreased mitochondrial bioenergetics and expression of genes related to fatty acid oxidation (ie, peroxisome proliferator-activated receptor-gamma coactivator-1α, a driver of mitochondrial biogenesis, and carnitine palmitoyltransferase-1ß, facilitator of fatty acid uptake). A CB1/CB2 dual agonist with limited brain penetration, CB-13, corrected these parameters. AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, mediated the ability of CB-13 to rescue mitochondrial function. In fact, the ability of CB-13 to rescue fatty acid oxidation-related bioenergetics, as well as expression of proliferator-activated receptor-gamma coactivator-1α and carnitine palmitoyltransferase-1ß, was abolished by pharmacological inhibition of AMPK using compound C and shRNA knockdown of AMPKα1/α2, respectively. Interventions that target CB/AMPK signaling might represent a novel therapeutic approach to address the multifactorial problem of cardiovascular disease.

Evolution and assessment of colour patterns in stream-resident and anadromous male threespine stickleback Gasterosteus aculeatus from three regions.

We compared the colour patterns of free swimming, reproductively active male threespine stickleback Gasterosteus aculeatus of the anadromous and stream ecotypes from three geographically distinct regions. Consistent with the hypothesis of environmentally mediated selection, our results indicate ecologically replicated differences in G. aculeatus coloration between anadromous and stream-resident populations, and that G. aculeatus probably have the visual acuity to discriminate colour pattern differences between anadromous and stream-resident fish.

Astrocyte dysfunction in Alzheimer disease.

Astrocytes are glial cells that are distributed throughout the central nervous system in an arrangement optimal for chemical and physical interaction with neuronal synapses and brain blood supply vessels. Neurotransmission modulates astrocytic excitability by activating an array of cell surface receptors and transporter proteins, resulting in dynamic changes in intracellular Ca2+ or Na+ . Ionic and electrogenic astrocytic changes, in turn, drive vital cell nonautonomous effects supporting brain function, including regulation of synaptic activity, neuronal metabolism, and regional blood supply. Alzheimer disease (AD) is associated with aberrant oligomeric amyloid ß generation, which leads to extensive proliferation of astrocytes with a reactive phenotype and abnormal regulation of these processes. Astrocytic morphology, Ca2+ responses, extracellular K+ removal, glutamate transport, amyloid clearance, and energy metabolism are all affected in AD, resulting in a deleterious set of effects that includes glutamate excitotoxicity, impaired synaptic plasticity, reduced carbon delivery to neurons for oxidative phosphorylation, and dysregulated linkages between neuronal energy demand and regional blood supply. This review summarizes how astrocytes are affected in AD and describes how these changes are likely to influence brain function. © 2017 Wiley Periodicals, Inc.

The Break Up: Evaluation of an Anti-Smoking Educational Campaign for Lesbians, Gays, and Bisexuals in Los Angeles County.

Lesbian, gay, and bisexual (LGB) adults in the United States have a higher prevalence of smoking than their heterosexual counterparts. In 2013, the Los Angeles County Department of Public Health launched a social marketing and outreach campaign called Break Up to reduce the prevalence of smoking in LGB communities. Break Up was evaluated using cross-sectional, street-intercept surveys before and near the end of campaign. Surveys measured demographics, campaign awareness, and self-reported smoking-related outcomes. Bivariate statistics and logistic regression models were used to identify whether campaign awareness was associated with smoking-related outcomes. Calls by LGB persons to a smokers' helpline were also measured. Among those interviewed at endline, 32.7% reported Break Up awareness. Awareness was associated with thinking of quitting smoking and ever taking steps to quit but not with smoking cessation (defined as not smoking in the past 30 days among those who had smoked in the past 6 months). There was a 0.7% increase in the percentage of weekly calls by LGB persons to the helpline in the year after the campaign. Break Up reached about a third of its intended audience. The campaign was associated with smoking cessation precursors and may have led to an increase in helpline utilization, but there is no evidence it affected quit attempts. This study adds to the limited literature on tobacco programs for LGB persons and, as far as we know, is one of the first to evaluate tobacco-free social marketing in this important yet understudied population.

Peripheral and Cerebral Resistance Arteries in the Spontaneously Hypertensive Heart Failure Rat: Effects of Stilbenoid Polyphenols.

Hypertension is associated with aberrant structure and mechanical properties of resistance arteries. We determined the effects of resveratrol, a non-flavonoid polyphenol found in foods such as red grapes, and structurally-similar analogues (pterostilbene and gnetol) on systolic blood pressure (SBP) and resistance arteries from the spontaneously hypertensive heart failure (SHHF) rat. SBP was elevated in 17-week-old SHHF vs. Sprague-Dawley rats (normotensive control; 194 ± 3 vs. 142 ± 6 mmHg, p < 0.01) and was unaffected by resveratrol, pterostilbene, or gnetol (2.5 mg/kg/d). Geometry and mechanical properties of pressurized mesenteric resistance arteries and middle cerebral arteries were calculated from media and lumen dimensions measured at incremental intraluminal pressures. SHHF arteries exhibited remodeling which consisted of augmented media-to-lumen ratios, and this was attenuated by stilbenoid treatment. Compliance was significantly reduced in SHHF middle cerebral arteries but not mesenteric arteries vis-à-vis increased wall component stiffness; stilbenoid treatment failed to normalize compliance and wall component stiffness. Our data suggest that neither AMPK nor ERK mediate stilbenoid effects. In conclusion, we observed arterial bed-specific abnormalities, where mesenteric resistance arteries exhibited remodeling and cerebral arteries exhibited remodeling and stiffening. Resveratrol, pterostilbene, and gnetol exhibited similar abilities to attenuate vascular alterations.

Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase.

Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca(2+) produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.

Poly(ADP-ribose) polymerase-1 causes mitochondrial damage and neuron death mediated by Bnip3.

Excessive pathophysiological activity of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP1) causes neuron death in brain hypoxia/ischemia by inducing mitochondrial permeability transition and nuclear translocation of apoptosis-inducing factor (AIF). Bcl-2/adenovirus E1B 19 kDa-interacting protein (Bnip3) is a prodeath BH3-only Bcl-2 protein family member that is induced in hypoxia, and has effects on mitochondrial permeability and neuronal survival similar to those caused by PARP1 activation. We hypothesized that Bnip3 is a critical mediator of PARP1-induced mitochondrial dysfunction and neuron death. Hypoxic death of mouse cortical neuron cultures was mitigated by deletion of either PARP1 or Bnip3, indicating that both factors are involved. Direct normoxic PARP1 activation by a DNA alkylating agent enhanced Bnip3 expression, and caused Bnip3-dependent mitochondrial membrane permeability, AIF translocation, and neuron death. Hypoxia produced PARP1-dependent depletion of nicotinamide adenine dinucleotide (NAD(+)) and inhibition of the NAD(+)-dependent class III histone deactelyase (HDAC) sirtuin-1 (SIRT1). This, in turn, led to hyperacetylation and nuclear localization of the forkhead box (Fox) protein FoxO3a, followed by enhanced association of FoxO3a with the Bnip3 upstream promoter region, increased levels of Bnip3 transcript, and elevated mitochondrial Bnip3 immunoreactivity. Finally, FoxO3a silencing using a lentiviral short hairpin RNA approach significantly reduced hypoxic Bnip3 expression, mitochondrial damage, and neuron death. Together, these data illustrate a direct PARP1-mediated hypoxic signaling pathway involving NAD(+) depletion, SIRT1 inhibition, FoxO3a-driven Bnip3 generation, and mitochondrial AIF release.

NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1.

BACKGROUND: The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation. METHODS: Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-κB transcriptional activity, DNA damage assesment and cytokine relesease profiling. RESULTS: TNFα induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNFα-induced transcriptional activation of NF-κB requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNFα was blocked in Ca(2+)-free medium, by Ca(2+) chelation with BAPTA-AM, and by D609, an inhibitor of phoshatidyl choline-specific phospholipase C (PC-PLC), but not by thapsigargin or by U73112, an inhibitor of phosphatidyl inisitol-specific PLC (PI -PLC). A TNFR1 blocking antibody reduced Ca(2+) influx and PARP-1 activation. TNFα-induced PARP-1 activation was also blocked by siRNA downregulation of ERK2 and by PD98059, an inhibitor of the MEK / ERK protein kinase cascade. Moreover, TNFα-induced NF-κB (p65) transcriptional activation was absent in cells expressing PARP-1 that lacked ERK2 phosphorylation sites, while basal NF-κB transcriptional activation increased in cells expressing PARP-1 with a phosphomimetic substitution at an ERK2 phophorylation site. CONCLUSIONS: These results suggest that TNFα induces PARP-1 activation through a signaling pathway involving TNFR1, Ca(2+) influx, activation of PC-PLC, and activation of the MEK1 / ERK2 protein kinase cascade. TNFα-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.

A National Asian-Language Smokers' Quitline--United States, 2012-2014.

INTRODUCTION: Until recently, in-language telephone quitline services for smokers who speak Asian languages were available only in California. In 2012, the Centers for Disease Control and Prevention (CDC) funded the national Asian Smokers' Quitline (ASQ) to expand this service to all states. The objective of this study was to examine characteristics of ASQ callers, how they heard about the quitline, and their use of the service. METHODS: Characteristics of callers from August 2012 through July 2014 were examined by using descriptive statistics. We examined demographics, cigarette smoking status, time to first cigarette, how callers heard about the quitline, and service use (receipt of counseling and medication) by using ASQ intake and administrative data. We analyzed these data by language and state. RESULTS: In 2 years, 5,771 callers from 48 states completed intake; 31% were Chinese (Cantonese or Mandarin), 38% were Korean, and 31% were Vietnamese. More than 95% of all callers who used tobacco were current daily cigarette smokers at intake. About 87% of ASQ callers were male, 57% were aged 45 to 64 years, 48% were uninsured, and educational attainment varied. Most callers (54%) were referred by newspapers or magazines. Nearly all eligible callers (99%) received nicotine patches. About 85% of smokers enrolled in counseling; counseled smokers completed an average of 4 sessions. CONCLUSION: ASQ reached Chinese, Korean, and Vietnamese speakers nationwide. Callers were referred by the promotional avenues employed by ASQ, and most received services (medication, counseling, or both). State quitlines and local organizations should consider transferring callers and promoting ASQ to increase access to cessation services.

Poly(ADP-ribose) polymerase 2 contributes to neuroinflammation and neurological dysfunction in mouse experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis characterized by entry of activated T cells and antigen presenting cells into the central nervous system and subsequent autoimmune destruction of nerve myelin. Previous studies revealed that non-selective inhibition of poly(ADP-ribose) polymerases (PARPs) 1 and 2 protect against neuroinflammation and motor dysfunction associated with EAE, but the role of the PARP-2 isoform has not yet been investigated selectively. RESULTS: EAE was induced in mice lacking PARP-2, and neurological EAE signs, blood-spine barrier (BSB) permeability, demyelination and inflammatory infiltration were monitored for 35 days after immunization. Mice lacking PARP-2 exhibited significantly reduced overall disease burden and peak neurological dysfunction. PARP-2 deletion also significantly delayed EAE onset and reduced BSB permeability, demyelination and central nervous system (CNS) markers of proinflammatory Th1 and Th17 T helper lymphocytes. CONCLUSIONS: This study represents the first description of a significant role for PARP-2 in neuroinflammation and neurological dysfunction in EAE.

Laparoscopic Graham Patch for Anterior Duodenal Perforation in a 6-Year-Old.

Peptic ulcer disease causing perforation is extremely rare in children and primarily affects teenagers. We present a case of a perforated peptic ulcer in a 6-year-old with abdominal pain and emesis with CT findings of moderate pneumoperitoneum and pelvic free fluid without a distinct cause. He was emergently transferred, found to be peritonitic, and taken to the operating room for diagnostic laparoscopy revealing an anterior duodenal ulcer, and underwent laparoscopic Graham patch repair. Postoperatively, the child had positive fecal antigen for H. pylori. He was treated with triple therapy and underwent subsequent testing to confirm eradication. Perforated peptic ulcer is an uncommon pediatric surgical problem, and imaging may not be diagnostic as in the case presented here. Thus, clinicians need to maintain a high index of suspicion when evaluating children with free air and a surgical abdomen in the setting of long-standing abdominal pain.

Ultrastructural analysis of throat dermal tissue and chromatophore components in the threespine stickleback (Gasterosteus aculeatus).

The threespine stickleback (Gasterosteus aculeatus) is an important model for studying the evolution of nuptial coloration, but histological analyses of color are largely lacking. Previous analyses of one nuptial coloration trait, orange-red coloration along the body, have indicated carotenoids are the main pigment producing this color. In addition, recent gene expression studies found variation in the correlates of throat coloration between the sexes and between populations, raising the possibility of variation in the mechanisms underlying superficially similar coloration. We used transmission electron microscopy (TEM) to investigate the histological correlates of color in the throat dermal tissue of threespine stickleback from Western North America, within and between sexes, populations, and ecotypes. Ultrastructural analysis revealed carotenoid-containing erythrophores to be the main chromatophore component associated with orange-red coloration in both males and females across populations. In individuals where some darkening of the throat tissue was present, with no obvious orange-red coloration, erythrophores were not detected. Melanophore presence was more population-specific in expression, including being the only chromatophore component detected in a population of darker fish. We found no dermal chromatophore units within colorless throat tissue. This work confirms the importance of carotenoids and the erythrophore in producing orange-red coloration across sexes, as well as melanin within the melanophore in producing darkened coloration, but does not reveal broad histological differences among populations with similar coloration.

Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1- variant No.11) and verified their efficiencies on the classic genome site EMX1-1, EMX1-1-OT1, and EMX1-1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an "enhanced specificity" SpCas9 variant released in 2016), on two genome sites, EMX1-1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.

Compensatory cross-talk between autophagy and glycolysis regulates senescence and stemness in heterogeneous glioblastoma tumor subpopulations.

Despite tremendous research efforts, successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Here, by using clinically-relevant patient-derived glioblastoma (GBM) cell models, we explore the cross-talk between glycolysis, autophagy, and senescence in maintaining tumor stemness. We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, bioinformatics analysis also revealed that the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, as evidenced by increased ß-galactosidase staining and upregulation of the cell cycle regulators p21Waf1/Cip1/CDKN1A and p16INK4A/CDKN2A, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Using various techniques including autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or inducing apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress.