Integrating Community Health Workers' Dual Clinic-Community Role in Safety-Net Primary Care: Implementation Lessons from a Pragmatic Diabetes-Prevention Trial.

BACKGROUND: Over a third of US adults carry a diagnosis of prediabetes, 70% of whom may progress to type 2 diabetes mellitus ("diabetes"). Community health workers (CHWs) can help patients undertake healthy behavior to prevent diabetes. However, there is limited guidance to integrate CHWs in primary care, specifically to address CHWs' dual clinic-based and community-oriented role. OBJECTIVE: Using evidence from CHWs' adaptations of a diabetes-prevention intervention in safety-net hospitals in New York City, we examine the nature, intent, and possible consequences of CHWs' actions on program fidelity. We propose strategies for integrating CHWs in primary care. DESIGN: Case study drawing on the Model for Adaptation Design and Impact (MADI) to analyze CHWs' actions during implementation of CHORD (Community Health Outreach to Reduce Diabetes), a cluster-randomized pragmatic trial (2017-2022) at Manhattan VA and Bellevue Hospital. PARTICIPANTS: CHWs and clinicians in the CHORD study, with a focus in this analysis on CHWs. APPROACH: Semi-structured interviews and focus group discussion with CHWs (n=4); semi-structured interviews with clinicians (n=17). Interpretivist approach to explain CHWs' adaptations using a mix of inductive and deductive analysis. KEY RESULTS: CHWs' adaptations extended the intervention in three ways: by extending social assistance, healthcare access, and operational tasks. The adaptations were intended to improve fit, reach, and retention, but likely had ripple effects on implementation outcomes. CHWs' focus on patients' complex social needs could divert them from judiciously managing their caseload. CONCLUSIONS: CHWs' community knowledge can support patient engagement, but overextension of social assistance may detract from protocolized health-coaching goals. CHW programs in primary care should explicitly delineate CHWs' non-health support to patients, include multiprofessional teams or partnerships with community-based organizations, establish formal communication between CHWs and clinicians, and institute mechanisms to review and iterate CHWs' work to resolve challenges in their community-oriented role.

Favorable Parental Perception of Proactive Nutrition Intervention in High-risk Pediatric Brain Tumor Population and Impact on Nutrition Outcomes.

Children with certain brain tumors often present with malnutrition and experience a decline in nutritional status throughout treatment. This can negatively affect outcomes. Studies have demonstrated that proactive enteral feeding can be beneficial to childhood cancer patients in helping to maintain or improve their nutritional status. To date, no classification parameters exist for pediatric brain tumor diagnoses and their corresponding nutritional risk. Our neuro-oncology team set out to develop a nutrition risk classification for pediatric brain tumors with a corresponding decision aid for nutrition intervention. We report the use of this decision aid in 15 pediatric brain tumor patients at high risk for nutritional deficits. Despite being high risk, weight loss did not exceed 5% in 93% (14/15) and 87% (13/15) of our patients from diagnosis to start of cycle 2 of chemotherapy and from diagnosis to end of therapy, respectively. Patients underweight at diagnosis (5/15) experienced improvements in nutritional status, and only 1 patient had a negative change in body mass index z-score ≥1 SD from diagnosis to end of therapy. This strategy was well-accepted by parents who reported satisfaction with the approach, their child's nutritional status throughout treatment, and the psychosocial aspects of feeding.

Transitioning from heparin to saline locks for central venous access devices in oncology: An evidence-based practice approach.

Central vascular access devices (CVADs) are often essential to the care of patients undergoing long-term cancer treatment. CVAD maintenance is an essential oncology nurse competency. Evidence-based practice (EBP) in flushing and locking help to prevent intraluminal occlusion, a common complication. Heparinized saline (HS) has been the standard locking solution for CVADs. However, research indicates no superiority of HS over normal saline (NS). The objectives of this EBP project were 1) to evaluate whether a significant difference in intraluminal occlusion was associated with the change from HS to NS use for locking CVADs in ambulatory oncology care, and 2) to evaluate the effects of peer nurse mentoring on nurses' and patients' perspectives about the practice change. Analysis of data revealed decreases in alteplase usage after transitioning to NS locking. Patient and nurse surveys indicated that peer nurse mentoring increased nurse and patient confidence and competence in making the practice transition.

Effects of anionic supplement source in prepartum negative dietary cation-anion difference diets on serum calcium, feed intake, and lactational performance of multiparous dairy cows.

Incidence of subclinical hypocalcemia in early postpartum dairy cows continues to be an animal welfare concern and an economic burden for producers. Feeding prepartum negative dietary cation-anion difference (DCAD) diets produces metabolic acidosis, which supports mobilization of bone calcium and reduces the incidence of hypocalcemia. Achieving a sufficient degree of metabolic acidosis without reducing dry matter intake (DMI) can be difficult. This study compared the ability of MegAnion (MA; Origination O2D Inc., Maplewood, MN), a new DCAD supplement designed to be more palatable than typical anionic salt sources, and another palatable commercial DCAD product, SoyChlor (SC; Landus Cooperative, Ralston, IA), to reduce urine pH (a surrogate for metabolic acidosis) without reducing prepartum DMI. A secondary objective was to assess the effect of these anionic supplements on postpartum serum calcium concentrations and DMI. Prepartum multiparous Holstein (HO) and crossbred (XX) cows were blocked by breed and expected calving date and randomly assigned within breed to total mixed rations (TMR) with MA or SC and DCAD values of -215 mEq/kg of DM. Cows (n = 56; 15 MA-HO, 12 SC-HO, 15 MA-XX, 14 SC-XX) consumed the treatment TMR for at least 19 d and completed the 28 d in milk (DIM) phase of the study. Urine and blood samples were collected weekly and at 1, 2, and 3 DIM. Data were analyzed as a randomized block design by repeated measures with week or DIM as the repeated effect. Prepartum urine pH decreased from 8.15 ± 0.27 before treatment to 6.12 ± 0.14 during treatment, was not affected by anionic supplement, and increased immediately after calving when all cows consumed the same early-lactation TMR. Prepartum serum calcium concentrations were not affected (2.34 vs. 2.33 ± 0.02 mmol/L) by treatment, whereas nonesterified fatty acids were lower (86 vs. 120 ± 10 mmol/L) and insulin was greater (215 vs. 174 ± 10 pmol/L) in cows fed MA than in cows fed SC. These differences are supported by the numerically greater prepartum DMI (1.2 kg/d) and energy balance (1.8 Mcal/d) of cows fed MA. However, pre- and postpartum DMI and other production variables, including body weight, body condition score, milk yield, and energy balance, were not affected by treatment. This lack of difference indicates that MA provides another effective source of anionic salts for diets designed to reduce urine pH and induce metabolic acidosis in prepartum dairy cows.

Cresyl violet: a superior fluorescent lysosomal marker.

We have characterized cresyl violet as a membrane-permeant fluorophore that localizes to lysosomes and acidic vacuoles of budding yeast, Drosophila, human, murine and canine cells. An acidotropic weak base, cresyl violet is shown to be virtually insensitive to physiological alkali and divalent cations. Because of its unique spectral properties, it can be used in combination with green, red and far-red fluorophores, is less susceptible to photobleaching than alternative acidotropic probes, and does not undergo photoconversion. At concentrations that yield bright labeling of acidic compartments, cresyl violet does not alter the organellar pH nor does it affect the buffering capacity. Its affordability, together with its chemical and spectral properties, make cresyl violet a superior lysosomal marker devoid of many of the negative characteristics associated with other lysosomal probes.

V-ATPases Containing a3 Subunit Play a Direct Role in Enamel Development in Mice.

Vacuolar H+ -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by µCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc.

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel NaV1.7.

Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

Suppression of mTORC1 activation in acid-α-glucosidase-deficient cells and mice is ameliorated by leucine supplementation.

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

The effect of chronic, non-pathogenic maternal immune activation on offspring postnatal muscle and immune outcomes.

The objective was to determine the effects of maternal inflammation on offspring muscle development and postnatal innate immune response. Sixteen first-parity gilts were randomly allotted to repeated intravenous injections with lipopolysaccharide (LPS; n = 8, treatment code INFLAM) or comparable volume of phosphate buffered saline (CON, n = 8). Injections took place every other day from gestational day (GD) 70 to GD 84 with an initial dose of 10 µg LPS/kg body weight (BW) increasing by 12% each time to prevent endotoxin tolerance. On GD 70, 76, and 84, blood was collected at 0 and 4 h postinjection via jugular or ear venipuncture to determine tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß concentrations. After farrowing, litter mortality was recorded, and the pig closest to litter BW average was used for dissection and muscle fiber characterization. On weaning (postnatal day [PND] 21), pigs were weighed individually and 2 barrows closest to litter BW average were selected for another study. The third barrow closest to litter BW average was selected for the postnatal LPS challenge. On PND 52, pigs were given 5 µg LPS/kg BW via intraperitoneal injection, and blood was collected at 0, 4, and 8 h postinjection to determine TNF-α concentration. INFLAM gilt TNF-α concentration increased (P < 0.01) 4 h postinjection compared to 0 h postinjection, while CON gilt TNF-α concentration did not differ between time points. INFLAM gilt IL-6 and IL-1ß concentrations increased (P = 0.03) 4 h postinjection compared to 0 h postinjection on GD 70, but did not differ between time points on GD 76 and 84. There were no differences between INFLAM and CON gilts litter mortality outcomes (P ≥ 0.13), but INFLAM pigs were smaller (P = 0.04) at birth and tended (P = 0.09) to be smaller at weaning. Muscle and organ weights did not differ (P ≥ 0.17) between treatments, with the exception of semitendinosus, which was smaller (P < 0.01) in INFLAM pigs. INFLAM pigs tended (P = 0.06) to have larger type I fibers. INFLAM pig TNF-α concentration did not differ across time, while CON pig TNF-α concentration peaked (P = 0.01) 4 h postinjection. TNF-α concentration did not differ between treatments at 0 and 8 h postinjection, but CON pigs had increased (P = 0.01) TNF-α compared to INFLAM pigs 4 h postinjection. Overall, maternal immune activation did not alter pig muscle development, but resulted in suppressed innate immune activation.

Maternal inflammation or immune activation impacts fetal development and subsequently the offspring's postnatal performance. In particular, maternal immune activation may be detrimental to fetal muscle development and alter postnatal immune responses, both of which are vital in determining livestock efficiency. However, understanding the relationship between maternal immune activation and offspring development is difficult as many models use a live pathogen. This introduces many confounding factors, including increased mortality, persistent postnatal infection, and potential copathogens. Therefore, the objective of this study was to determine the effect of maternal inflammation on offspring muscle development and postnatal inflammatory response using repeated injections of a nonpathogenic immune stimulant. Each injection successfully induced an inflammatory response as indicated by increased rectal temperature and circulating inflammatory markers. The gestational challenge did not result in increased litter mortality. Further, muscle development was not altered in piglets exposed to gestational inflammation. However, when challenged with the same immune stimulant given to the dams, pigs exposed to maternal inflammation had a remarkably suppressed immune response compared to controls. Overall, maternal inflammation independent of infection affected offspring immune function, but not muscle development.

Characterizing sow feed intake during lactation to explain litter and subsequent farrowing performance.

Variation in feed intake results in nearly 20% of sows consuming less than the recommended lysine (Lys) intake for lactating sows. The Lys requirement for lactating sows is based on litter size and piglet average daily gain which influences milk production. Litter size continues to increase every year causing the need for routine reevaluation of nutrient requirements. If dietary inclusion levels are not continuously adjusted this can lead to inadequate daily Lys and energy intake and may negatively impact sow body condition and litter performance. The objective was to characterize the average daily feed intake (ADFI) of sows and define feed intake patterns and their effects on sow body weight, farrowing performance, litter performance, and subsequent farrowing performance. ADFI during lactation was recorded for 4,248 sows from 7 independent research studies. Data collection occurred from November 2021 through November 2023 at a commercial breed-to-wean facility in western Illinois. Each sow was categorized as: consistently low intake (< 5.5 kg/d) throughout the lactation (LLL); low intakes (< 5 kg/d) in the first week, then gradually increased throughout the rest of the lactation period (LHH); gradual increase in intake throughout lactation with no drop and a peak intake after day 10 of lactation (gradual); rapid increase in intake with no drop and the peak intake met before day 10 (rapid); a major drop in feed intake (> 1.6 kg decrease for ≥ 2 d) any time during lactation (MAJOR); minor drop (≤ 1.6 kg for ≥ 2 d; MINOR). Sows were also separated into low (quartile 1; ≤ 25%), average (quartile 2 through 3), or high feed intake (quartile 4; ≥ 75%) by parity (P1, P2, P3+). Sows in the LLL category were younger in parity, had the greatest preweaned mortality, weaned the lightest average pigs, and experienced the greatest loss in body weight percentage compared with sows in all other feed intake categories. Furthermore, sows in the LLL and LHH categories had one fewer subsequent pig born compared with sows in the other four categories. These data support historical findings that feed intake patterns directly contribute to current litter farrowing performance. Lactation intake patterns also influence subsequent farrowing performance. Identifying under-consuming sows that are likely Lys and energy deficient allows producers opportunities to promote consistent, adequate daily intakes to these groups and mitigate negative impacts on sow and litter performance.

This study investigated different sow feed intake patterns during lactation and average daily feed intakes within parity on current and subsequent farrowing and litter performance. Findings revealed sows that have consistently low intake throughout the lactation period have a significant reduction in average pig wean weight, a greater percentage of pre-wean mortality, and take an additional day or longer to return to estrus compared with sows that have average or above feed intake throughout the lactation period. Specifically, older parity sows were heavier, had greater feed intake, nursed heavier litters, and had litters with less preweaned mortality compared with younger parity sows. The average pig weaned weight and subsequent total pigs born improved as intake increased within parity. Prewean mortality decreased as feed intake increased within parity. These findings highlight the importance of ensuring sows are not only eating enough, but that they are consuming more than average when possible, to continually improve current and subsequent farrowing and litter performance. This study provides important information that will allow producers to target specific under-consuming sows and then promote consistent and high daily lactation intakes. Targeting these potentially nutrient-restricted sows may help reduce negative impacts on sow and litter performance.

Riding the Wave: Reactive Vector-Borne Disease Policy Renders the United States Vulnerable to Outbreaks and Insecticide Resistance.

Funding for vector-borne disease surveillance, management, and research is cyclical and reactive in the United States. The subsequent effects have yielded gross inequities nationally that unintentionally support recurrent outbreaks. This policy forum is comprised of four primary subsections that collectively identify specific areas for improvement and offer innovative solutions to address national inadequacies in vector borne disease policy and infrastructure.

Needs Assessment of Southeastern United States Vector Control Agencies: Capacity Improvement Is Greatly Needed to Prevent the Next Vector-Borne Disease Outbreak.

A national 2017 vector control capacity survey was conducted to assess the United States' (U.S.'s) ability to prevent emerging vector-borne disease. Since that survey, the southeastern U.S. has experienced continued autochthonous exotic vector-borne disease transmission and establishment of invasive vector species. To understand the current gaps in control programs and establish a baseline to evaluate future vector control efforts for this vulnerable region, a focused needs assessment survey was conducted in early 2020. The southeastern U.S. region was targeted, as this region has a high probability of novel vector-borne disease introduction. Paper copies delivered in handwritten envelopes and electronic copies of the survey were delivered to 386 unique contacts, and 150 returned surveys were received, corresponding to a 39% response rate. Overall, the survey found vector control programs serving areas with over 100,000 residents and those affiliated with public health departments had more core capabilities compared to smaller programs and those not affiliated with public health departments. Furthermore, the majority of vector control programs in this region do not routinely monitor for pesticide resistance. Taken as a whole, these results suggest that the majority of the southeastern U.S. is vulnerable to vector-borne disease outbreaks. Results from this survey raise attention to the critical need of providing increased resources to bring all vector control programs to a competent level, ensuring that public health is protected from the threat of vector-borne disease.

A Systematic Review and Analysis of the Use of Polygenic Scores in Pharmacogenomics.

Polygenic scores (PGSs) have emerged as promising tools for complex trait risk prediction. The application of these scores to pharmacogenomics provides new opportunities to improve the prediction of treatment outcomes. To gain insight into this area of research, we conducted a systematic review and accompanying analysis. This review uncovered 51 papers examining the use of PGSs for drug-related outcomes, with the majority of these papers focusing on the treatment of psychiatric disorders (n = 30). Due to difficulties in collecting large cohorts of uniformly treated patients, the majority of pharmacogenomic PGSs were derived from large-scale genome-wide association studies of disease phenotypes that were related to the pharmacogenomic phenotypes under investigation (e.g., schizophrenia-derived PGSs for antipsychotic response prediction). Examination of the research participants included in these studies revealed that the majority of cohort participants were of European descent (78.4%). These biases were also reflected in research affiliations, which were heavily weighted towards institutions located in Europe and North America, with no first or last authors originating from institutions in Africa or South Asia. There was also substantial variability in the methods used to develop PGSs, with between 3 and 6.6 million variants included in the PGSs. Finally, we observed significant inconsistencies in the reporting of PGS analyses and results, particularly in terms of risk model development and application, coupled with a lack of data transparency and availability, with only three pharmacogenomics PGSs deposited on the Polygenic Score Catalog. These findings highlight current gaps and key areas for future pharmacogenomic PGS research.

Cytosolic H+ microdomain developed around AE1 during AE1-mediated Cl-/HCO3- exchange.

Microdomains, regions of discontinuous cytosolic solute concentration enhanced by rapid solute transport and slow diffusion rates, have many cellular roles. pH-regulatory membrane transporters, like the Cl−/HCO3− exchanger AE1, could develop H+ microdomains since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. We examined whether the pH environment surrounding AE1 differs from other cellular locations. As AE1 drives Cl−/HCO3− exchange, differences in pH, near and remote from AE1, were monitored by confocal microscopy using two pH-sensitive fluorescent proteins: deGFP4 (GFP) and mNectarine (mNect). Plasma membrane (PM) pH (defined as ∼1 µm region around the cell periphery) was monitored by GFP fused to AE1 (GFP.AE1), and mNect fused to an inactive mutant of the Na+-coupled nucleoside co-transporter, hCNT3 (mNect.hCNT3). GFP.AE1 to mNect.hCNT3 distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the GFP.AE1­mNect.hCNT3 distance increased, mNect.hCNT3 detected the Cl−/HCO3− exchange-associated cytosolic pH change with a time delay and reduced rate of pH change compared to GFP.AE1. We found that a H+ microdomain 0.3 µm in diameter forms around GFP.AE1 during physiological HCO3− transport. Carbonic anhydrase isoform II inhibition prevented H+ microdomain formation. We also measured the rate of H+ movement from PM GFP.AE1 to endoplasmic reticulum (ER), using mNect fused to the cytosolic face of ER-resident calnexin (CNX.mNect). The rate of H+ diffusion through cytosol was 60-fold faster than along the cytosolic surface of the plasma membrane. The pH environment surrounding pH regulatory transport proteins may differ as a result of H+ microdomain formation, which will affect nearby pH-sensitive processes.

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

Prevalence and predictors of weight loss during induction therapy for childhood acute lymphoblastic leukemia.

OBJECTIVE: Children with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma often experience significant weight gain during induction therapy. However, a subgroup of patients may experience weight loss, which can impact outcomes; thus, identifying and understanding this underrecognized concern is important. Our aim was to identify the prevalence and predictors for weight loss during ALL induction therapy. METHODS: This was a single-institution retrospective study of 187 patients, ages 2 to 20 y, diagnosed with ALL or lymphoblastic lymphoma. We analyzed weight trends during induction therapy and predictors of weight loss. RESULTS: Significant weight loss (≥5%) occurred in 17% of patients. Having high-risk disease, trisomy 21, overweight/obese status at the time of diagnosis, and/or hyperglycemia were positively associated with weight loss and negatively associated with weight gain during induction therapy. CONCLUSION: Future studies should aim to better understand the etiology and importance of weight loss during induction therapy.

Red fluorescent protein pH biosensor to detect concentrative nucleoside transport.

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H(+)-coupled nucleoside transport, pH 5.5, under both Na(+)-free and Na(+)-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H(+)-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+ (1.09 +/- 0.11 or 1.18 +/- 0.32 mm min(-1), respectively). This suggests that in acidic Na(+)-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na(+)-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H(+)-coupled transport proteins.