Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells.

Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch1 expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.

Single-cell intracellular pH dynamics regulate the cell cycle by timing the G1 exit and G2 transition.

Transient changes in intracellular pH (pHi) regulate normal cell behaviors, but roles for spatiotemporal pHi dynamics in single-cell behaviors remain unclear. Here, we mapped single-cell spatiotemporal pHi dynamics during mammalian cell cycle progression both with and without cell cycle synchronization. We found that single-cell pHi is dynamic throughout the cell cycle: pHi decreases at G1/S, increases in mid-S, decreases at late S, increases at G2/M and rapidly decreases during mitosis. Importantly, although pHi is highly dynamic in dividing cells, non-dividing cells have attenuated pHi dynamics. Using two independent pHi manipulation methods, we found that low pHi inhibits completion of S phase whereas high pHi promotes both S/G2 and G2/M transitions. Our data also suggest that low pHi cues G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. Furthermore, dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work reveals that spatiotemporal pHi dynamics are necessary for cell cycle progression at multiple phase transitions in single human cells.

SGLT2-independent effects of canagliflozin on NHE3 and mitochondrial complex I activity inhibit proximal tubule fluid transport and albumin uptake.

Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo.NEW & NOTEWORTHY Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells.

Preconception Periodontitis and Risk of Spontaneous Abortion in a Prospective Cohort Study.

Few studies have evaluated the association between periodontitis and spontaneous abortion (SAB), and all had limitations. We used data from the Pregnancy Study Online (PRESTO), a prospective preconception cohort study of 3,444 pregnancy planners in the United States and Canada (2019-2022), to address this question. Participants provided self-reported data on periodontitis diagnosis, treatment, and symptoms of severity (i.e., loose teeth) via the enrollment questionnaire. SAB (pregnancy loss at <20 weeks' gestation) was assessed via bimonthly follow-up questionnaires. Participants contributed person-time from the date of a positive pregnancy test to the gestational week of SAB, loss to follow-up, or 20 weeks' gestation, whichever came first. We fitted Cox regression models with weeks of gestation as the time scale to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs), and we used inverse probability of treatment weighting to account for differential loss to follow-up. We used probabilistic quantitative bias analysis to estimate the magnitude and direction of the effect of exposure misclassification bias on results. In weighted multivariable models, we saw no appreciable association between preconception periodontitis diagnosis (HR = 0.97, 95% CI: 0.76, 1.23) or treatment (HR = 1.01, 95% CI: 0.79, 1.27) and SAB. A history of loose teeth was positively associated with SAB (HR = 1.38, 95% CI: 0.88, 2.14). Quantitative bias analysis indicated that our findings were biased towards the null but with considerable uncertainty in the bias-adjusted results.

Sexual dysfunction, distress, and care-seeking among females during the preconception period.

BACKGROUND: Despite the high prevalence of female sexual dysfunction in population-based studies and the importance of sexual functioning for mixed-sex couples attempting conception, little is known about female sexual function in the preconception period. OBJECTIVE: This descriptive study aimed to assess the prevalence of female sexual dysfunction, distress, and pain with intercourse in a preconception population of pregnancy planners. The study also explored the extent to which participants discussed their sex lives with a healthcare provider during a preconception visit. STUDY DESIGN: We used data from Pregnancy Study Online, a web-based preconception cohort study (August 2020-October 2022). Eligible participants identified as female and were aged 21 to 45 years, residents of the United States or Canada, attempting pregnancy, and not using fertility treatments at cohort entry. At enrollment, participants completed a detailed baseline questionnaire. Thirty days after enrollment, participants were invited to complete an optional questionnaire about sexual function. Our study included 1120 participants who responded to the sexual function questionnaire within 1 year of completing their baseline questionnaire. We assessed sexual dysfunction using the 6-item Female Sexual Function Index, and sexual distress using the Female Sexual Distress Scale, which assess sexual function and distress in the previous 4 weeks, respectively. We also asked participants whether they had discussed their plans to conceive with a healthcare provider, and if so, whether they discussed their sex lives. If not, we collected information on perceived barriers. RESULTS: Twenty-five percent of the sample met criteria for female sexual dysfunction, whereas 12.2% met the criteria for sexual distress; 8% of our sample reported both sexual dysfunction and sexual distress. Thirty percent reported at least some pain with intercourse in the past 4 weeks. Although over 80% of the sample reported discussing their conception plans with a healthcare provider, 70% of these participants did not discuss their sex lives. The most commonly reported reasons for not discussing their sex life with a provider was not experiencing a sexual health issue, the provider not asking, feeling nervous/uncomfortable/ashamed, and feeling it was not relevant to becoming pregnant or inappropriate to discuss. The percentage of participants who reported discussing their sex lives varied across provider type, with those seeing midwives having the highest percentage (39%), followed by nurse practitioners (36%) and obstetrician-gynecologists (34%). CONCLUSION: Sexual dysfunction, distress, and painful intercourse are prevalent in the preconception period, but participants frequently did not discuss their sex lives when discussing plans to conceive. The provider not asking was a commonly reported barrier. Providers may consider raising the issue of sexual functioning at the time of a preconception visit to better support patients who may be dealing with a sexual function issue while attempting pregnancy. These findings may not generalize beyond a primarily non-Hispanic White, highly educated, and high-income population.

First-trimester surgical abortion practice in Canada in 2012.

OBJECTIVE: To evaluate practices among first-trimester surgical abortion facilities and providers in Canada in 2012 and examine the characteristics of the surgical abortion work force. DESIGN: Self-administered paper or electronic survey adapted from a survey previously fielded in the United States. SETTING: Canada. PARTICIPANTS: Facility administrators and physicians. MAIN OUTCOMES MEASURES: Descriptive statistics on reported first-trimester surgical abortion practice and provider demographic characteristics. RESULTS: Eighty-three percent of identified facilities (78 of 94) and 178 physicians responded. Of the respondents, 99% of facilities and 96% of physicians provided first-trimester surgical abortions. Responding facilities provided 68,154 first-trimester surgical abortions in 2012. This represented 96% of their reported total (combined medical and surgical) first-trimester abortions. More than half (55%) of responding facilities were community based, while 45% were hospital affiliated. Most physician providers were female (68%) and were family doctors (59%). Preoperatively, 96% of physicians routinely used ultrasound and 89% gave perioperative antibiotics. Almost half (48%) used manual vacuum aspiration, but less than 35% did so beyond 9 weeks after the last menstrual period. At most facilities, most procedures were performed under combined local anesthesia and intravenous sedation (73%); only 7% indicated deep sedation or general anesthesia were used exclusively. Postoperatively, 81% of physicians performed immediate tissue examination and 96% offered postabortion contraception on the same day as the abortion. Other assessed outcomes included medication regimens and cervical preparation, with a high degree of consistency among facilities and physicians. CONCLUSION: First-trimester surgical abortion providers are mostly family physicians and most are female. Practices across Canada were mostly uniform and followed evidence-based guidelines. Uptake of the most recent Canadian practice guidelines may help further standardize patient care and improve routine perioperative antibiotic use and immediate tissue examination.

Primary Care-Based Cardiovascular Disease Risk Management After Adverse Pregnancy Outcomes: a Narrative Review.

Several common adverse pregnancy outcomes can reveal subclinical or latent cardiovascular disease (CVD) risk, transiently exposed through the physiologic stress of pregnancy. The year after pregnancy may be a singular opportunity to identify and initiate treatment for CVD risk, even before the onset of traditional CVD risk factors. However, clinical guidance regarding CVD risk management after adverse pregnancy outcomes is lacking. We therefore conducted a systematic review of US clinical practice guidelines and professional society recommendations to inform primary care-based CVD risk management after adverse pregnancy outcomes. We identified 13 relevant publications. While most recommendations were based on limited or weak evidence, we identified several areas of consensus. First, individuals with an adverse pregnancy outcome associated with future CVD are likely to benefit from CVD risk assessment-accompanied by education, counseling, and support for lifestyle modification-beginning within the first postpartum year. Second, among clinicians, clear and consistent documentation about adverse pregnancy outcomes and recommended follow-up is important to coordinate care after pregnancy. In addition, patients need to be informed about their pregnancy complications and associated CVD risks, so that they can make informed health care and lifestyle decisions. Finally, in general, CVD prevention in the year after an adverse pregnancy outcome focuses on lifestyle modification, reserving pharmacotherapy for the highest-risk patients and those with traditional CVD risk factors. While postpartum lifestyle interventions show promise for reducing CVD risk after adverse pregnancy outcomes, continued research to determine the optimal content, timing, and long-term effects of such interventions is needed.

Predictors of Non-Response to a Sexual Health Survey in a North American Preconception Cohort Study.

BACKGROUND: Refusal to participate in studies related to sexual health can compromise the internal and external validity of findings. Research examining non-response to sexual health studies has primarily focused on predictors such as specific sexual attitudes and behaviors. AIM: Evaluate predictors of non-response to a supplemental sexual health survey added to a web-based cohort study, focusing on predictors that may be important in epidemiologic studies of sexual health. METHODS: In March 2021, we added the "Sexual Health and Wellbeing Questionnaire" (SQ), an optional supplemental sexual health survey, to the protocol for Pregnancy Study Online , a web-based North American prospective cohort study. Eligible participants identified as female and were aged 21-45 years, actively trying to conceive, and in a relationship with a male partner. Participants completed a baseline questionnaire at enrollment and follow-up questionnaires every 8 weeks. Participants were invited to complete the SQ 30 days after baseline questionnaire completion. The analytic sample included all Pregnancy Study Online participants who enrolled between March 2021 and December 2021 and was divided into 3 mutually-exclusive groups: (i) those who completed the baseline questionnaire only (ie, did not complete a follow-up questionnaire or the SQ), (ii) those who completed at least 1 follow-up questionnaire but not the SQ, and (iii) participants who completed the SQ (with or without a follow-up questionnaire). We compared sociodemographic, medical, lifestyle, and reproductive factors across these groups. RESULTS: Of the 1,491 enrolled participants, 302 (20.3%) completed the baseline questionnaire only, 259 (17.4%) completed a follow-up questionnaire but not the SQ, and 930 (62.4%) completed the SQ. Strong predictors of non-response (absolute difference in response >10% comparing SQ responders to baseline-only responders) included longer pregnancy attempt time at study entry, a history of infertility, and lower income and education. Compared with response to the follow-up questionnaire only, SQ response was lower among Hispanic/Latina participants and participants aged <25 years. CLINICAL TRANSLATION: The addition of sexual health surveys to established cohort studies may be an effective way to expand epidemiologic sex research efforts. STRENGTHS & LIMITATIONS: Study strengths include the prospective design, geographic heterogeneity of the cohort, and use of online methods. Our findings may not generalize to clinic-based sex research. CONCLUSION: We report that in an established North American cohort study, response to the SQ exceeded 60%. We observed few strong predictors for SQ non-response among engaged participants. Bond JC, Abrams J, Wesselink AK, et al. Predictors of Non-Response to a Sexual Health Survey in a North American Preconception Cohort Study. J Sex Med 2022;19:1707-1715.

Low pH Facilitates Heterodimerization of Mutant Isocitrate Dehydrogenase IDH1-R132H and Promotes Production of 2-Hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) is a key metabolic enzyme for maintaining cytosolic levels of α-ketoglutarate (AKG) and preserving the redox environment of the cytosol. Wild-type (WT) IDH1 converts isocitrate to AKG; however, mutant IDH1-R132H that is recurrent in human cancers catalyzes the neomorphic production of the oncometabolite d-2-hydroxyglutrate (D-2HG) from AKG. Recent work suggests that production of l-2-hydroxyglutarte in cancer cells can be regulated by environmental changes, including hypoxia and intracellular pH (pHi). However, it is unknown whether and how pHi affects the activity of IDH1-R132H. Here, we show that in cells IDH1-R132H can produce D-2HG in a pH-dependent manner with increased production at lower pHi. We also identify a molecular mechanism by which this pH sensitivity is achieved. We show that pH-dependent production of D-2HG is mediated by pH-dependent heterodimer formation between IDH1-WT and IDH1-R132H. In contrast, neither IDH1-WT nor IDH1-R132H homodimer formation is affected by pH. Our results demonstrate that robust production of D-2HG by IDH1-R132H relies on the coincidence of (1) the ability to form heterodimers with IDH1-WT and (2) low pHi or highly abundant AKG substrate. These data suggest cancer-associated IDH1-R132H may be sensitive to physiological or microenvironmental cues that lower pH, such as hypoxia or metabolic reprogramming. This work reveals new molecular considerations for targeted therapeutics and suggests potential synergistic effects of using catalytic IDH1 inhibitors targeting D-2HG production in combination with drugs targeting the tumor microenvironment.

An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics.

Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use nonspecific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes time scale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for up to 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to unstimulated ArchT cells as well as control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and single cell behaviors such as membrane ruffling. This tool will be transformative in facilitating experiments that are required to determine roles for increased pHi in driving single cell behaviors.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Intracellular pH dynamics and charge-changing somatic mutations in cancer.

An unresolved question critical for understanding cancer is how recurring somatic mutations are retained and how selective pressures drive retention. Increased intracellular pH (pHi) is common to most cancers and is an early event in cancer development. Recent work shows that recurrent somatic mutations can confer an adaptive gain in pH sensing to mutant proteins, enhancing tumorigenic phenotypes specifically at the increased pHi of cancer. Newly identified amino acid mutation signatures in cancer suggest charge-changing mutations define and shape the mutational landscape of cancer. Taken together, these results support a new perspective on the functional significance of somatic mutations in cancer. In this review, we explore existing data and new directions for better understanding how changes in dynamic pH sensing by somatic mutation might be conferring a fitness advantage to the high pH of cancer.

Cancer cell behaviors mediated by dysregulated pH dynamics at a glance.

Dysregulated pH is a common characteristic of cancer cells, as they have an increased intracellular pH (pHi) and a decreased extracellular pH (pHe) compared with normal cells. Recent work has expanded our knowledge of how dysregulated pH dynamics influences cancer cell behaviors, including proliferation, metastasis, metabolic adaptation and tumorigenesis. Emerging data suggest that the dysregulated pH of cancers enables these specific cell behaviors by altering the structure and function of selective pH-sensitive proteins, termed pH sensors. Recent findings also show that, by blocking pHi increases, cancer cell behaviors can be attenuated. This suggests ion transporter inhibition as an effective therapeutic approach, either singly or in combination with targeted therapies. In this Cell Science at a Glance article and accompanying poster, we highlight the interconnected roles of dysregulated pH dynamics in cancer initiation, progression and adaptation.

A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity.

The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi However, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2 binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2 binding than AXXA3. When expressed in cells, NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHi enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2 binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHi contributes to cell behaviors, most notably the biology of cancer cells.

Abortion health services in Canada: Results of a 2012 national survey.

OBJECTIVE: To determine the location of Canadian abortion services relative to where reproductive-age women reside, and the characteristics of abortion facilities and providers. DESIGN: An international survey was adapted for Canadian relevance. Public sources and professional networks were used to identify facilities. The bilingual survey was distributed by mail and e-mail from July to November 2013. SETTING: Canada. PARTICIPANTS: A total of 94 abortion facilities were identified. MAIN OUTCOME MEASURES: The number and location of services were compared with the distribution of reproductive-age women by location of residence. RESULTS: We identified 94 Canadian facilities providing abortion in 2012, with 48.9% in Quebec. The response rate was 83.0% (78 of 94). Facilities in every jurisdiction with services responded. In Quebec and British Columbia abortion services are nearly equally present in large urban centres and rural locations throughout the provinces; in other Canadian provinces services are chiefly located in large urban areas. No abortion services were identified in Prince Edward Island. Respondents reported provision of 75 650 abortions in 2012 (including 4.0% by medical abortion). Canadian facilities reported minimal or no harassment, in stark contrast to American facilities that responded to the same survey. CONCLUSION: Access to abortion services varies by region across Canada. Services are not equitably distributed in relation to the regions where reproductive-age women reside. British Columbia and Quebec have demonstrated effective strategies to address disparities. Health policy and service improvements have the potential to address current abortion access inequity in Canada. These measures include improved access to mifepristone for medical abortion; provincial policies to support abortion services; routine abortion training within family medicine residency programs; and increasing the scope of practice for nurses and midwives to include abortion provision.

First-trimester medical abortion practices in Canada: National survey.

OBJECTIVE: To understand the current availability and practice of first-trimester medical abortion (MA) in Canada. DESIGN: Using public sources and professional networks, abortion facilities across Canada were identified for a cross-sectional survey on medical and surgical abortion. English and French surveys were distributed by surface or electronic mail between July and November 2013. SETTING: Canada. PARTICIPANTS: A total of 94 abortion facilities were identified. MAIN OUTCOME MEASURES: Descriptive statistics on MA practice and facility and provider characteristics, as well as comparisons of MA practice by facility and provider characteristics using χ2 and t tests. RESULTS: A total of 78 of 94 (83.0%) facilities responded. Medical abortion represented 3.8% of first-trimester abortions reported (2706 of 70 860) in 2012. Among the facilities offering MA, 45.0% performed fewer than 500 first-trimester abortions a year, while 35.0% performed more than 1000. More MAs were performed in private offices or ambulatory health centres than in hospitals. Sixty-two physicians from 28 of 78 facilities reported providing first-trimester MA; 87.1% also provided surgical abortion. More than three-quarters of MA physicians were female and 56.5% were family physicians. A preponderance (85.2%) of providers offered methotrexate with misoprostol. Nearly all physicians (90.3%) required patients to have an ultrasound before MA, and 72.6% assessed the completion of the abortion with ultrasonography. Most physicians (74.2%) offered MA through 49 days after the onset of the last menstrual period, and 21.0% offered MA through 50 to 56 days; 37.1% reported providing MA to patients who lived more than 2 hours away. Four physicians from 1 site provided MA via telemedicine. CONCLUSION: In Canada, MA provision using methotrexate and misoprostol is consistent with best-practice guidelines, but MA is rare and its availability is unevenly distributed.

Intracellular pH regulates ß-catenin with low pHi increasing adhesion and signaling functions.

Intracellular pH (pHi) dynamics are linked to cell processes including proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein ß-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on ß-catenin abundance and functions have not been characterized. Here, we show that low pHi stabilizes ß-catenin in epithelial cells using population-level and single-cell assays. ß-catenin abundance is increased at low pHi and decreased at high pHi. We also assay single-cell protein degradation rates to show that ß-catenin half-life is longer at low compared to high pHi. Importantly, we show that AJs are not disrupted by ß-catenin loss at high pHi due to rescue by plakoglobin. Finally, we show that low pHi increases ß-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state. This work characterizes pHi as a rheostat regulating ß-catenin abundance, stability, and function and implicates ß-catenin as a molecular mediator of pHi-dependent cell processes.

Development and Evaluation of a Novel Approach to Patient-Centered Contraceptive Counseling.

OBJECTIVES: We aimed to develop and evaluate a novel model, PHI CARE, that provides a standardized framework for shared decision-making in contraceptive counseling. METHODS: We developed the PHI CARE model with national experts, piloted it at three family planning clinics, and finalized it following additional patient and clinician review. We recruited pregnancy-capable people for an evaluation study via simulated contraceptive counseling and identified salient themes through inductive and deductive coding. RESULTS: Participants (n = 12) felt that counseling with the PHI CARE model was an improvement over previous counseling experiences and led to feelings of empowerment; participants did not feel pressured to decide about method use, felt in control during the conversation, and appreciated the lack of assumptions about their desires around pregnancy. Despite the standardized format, participants felt the counseling was individualized, "values-based," and tailored to their preferences. CONCLUSION: PHI CARE is a model to support clinicians and counselors in operationalizing the principles of shared decision-making in contraceptive counseling. Through standardization, PHI CARE allows for a more individualized experience for patients and addresses many critiques of traditional counseling. PRACTICE IMPLICATIONS: PHI CARE is a memorable, brief tool that can be used for patient-centered contraceptive counseling in any clinical encounter.

Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through ß-catenin.

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize ß-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in ß-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.