TRPC4 and GIRK channels underlie neuronal coding of firing patterns that reflect Gq/11-Gi/o coincidence signals of variable strengths.

Transient receptor potential canonical 4 (TRPC4) is a receptor-operated cation channel codependent on both the Gq/11­phospholipase C signaling pathway and Gi/o proteins for activation. This makes TRPC4 an excellent coincidence sensor of neurotransmission through Gq/11- and Gi/o-coupled receptors. In whole-cell slice recordings of lateral septal neurons, TRPC4 mediates a strong depolarizing plateau that shuts down action potential firing, which may or may not be followed by a hyperpolarization that extends the firing pause to varying durations depending on the strength of Gi/o stimulation. We show that the depolarizing plateau is codependent on Gq/11-coupled group I metabotropic glutamate receptors and on Gi/o-coupled γ-aminobutyric acid type B receptors. The hyperpolarization is mediated by Gi/o activation of G protein­activated inwardly rectifying K+ (GIRK) channels. Moreover, the firing patterns, elicited by either electrical stimulation or receptor agonists, encode information about the relative strengths of Gq/11 and Gi/o inputs in the following fashion. Pure Gq/11 input produces weak depolarization accompanied by firing acceleration, whereas pure Gi/o input causes hyperpolarization that pauses firing. Although coincident Gq/11­Gi/o inputs also pause firing, the pause is preceded by a burst, and both the pause duration and firing recovery patterns reflect the relative strengths of Gq/11 versus Gi/o inputs. Computer simulations demonstrate that different combinations of TRPC4 and GIRK conductances are sufficient to produce the range of firing patterns observed experimentally. Thus, concurrent neurotransmission through the Gq/11 and Gi/o pathways is converted to discernible electrical responses by the joint actions of TRPC4 and GIRK for communication to downstream neurons.

A prospective phase II clinical trial/IDEAL Stage 2a series of single-port robotic colorectal surgery for abdominal and transanal cases.

AIM: Slow laparoscopy adoption accelerated the uptake of robotic surgery. However, the current robotic platforms have limitations in transanal applications and multiple port sites. The da Vinci single-port (SP) robot is currently used on trial for colorectal surgery, and broad assessment of outcomes is needed. We aimed to report findings of a phase II clinical trial of SP robotic colorectal surgery. METHODS: A sequentially reported prospective case series was performed on patients using SP robotics at a tertiary referral centre from 1 October 2018 to 31 August 2021. Cases were stratified into abdominal and transanal cohorts. Demographics, intra-operative variables and 30-day postoperative outcomes were evaluated. Univariate analysis was performed, with statistical process control for the docking process. Main outcomes were conversion rates, morbidity, mortality and point of standardization of docking. RESULTS: In all, 133 patients were included: 93 (69.92%) abdominal and 40 (30.08%) transanal. The main diagnosis was rectal cancer (n = 59) and the procedure performed a robotic transanal abdominal transanal radical proctosigmoidectomy (n = 30). There were no conversions to open surgery. Two abdominal (2.15%) and three transanal cases (7.50%) were converted to laparoscopy. All colorectal adenocarcinomas had negative margins, proper lymph node harvest and complete mesorectal excision, as appropriate. Docking became a standardized process at cases 34 (abdominal) and 23 (anorectal). After surgery, bowel function returned on mean day 2 (abdominal) and 1 (transanal). The morbidity rate was 15.05% (abdominal) and 27.50% (transanal). There were two major morbidities in each cohort. Overall, there were three (2.65%) readmissions, one reoperation and no mortality. CONCLUSIONS: Single-port robotics is feasible for all types of colorectal procedures, with good clinical and oncological outcomes. With this development in colorectal surgery, further studies can develop best practices with this novel technology.

Antibody-Free Quantification of Serum Chromogranin A by Targeted Mass Spectrometry.

BACKGROUND: Chromogranin A (CgA) is a 48 kDa protein that serves as a diagnostically sensitive, but nonspecific, serum biomarker for neuroendocrine tumors. Immunoassays for CgA are not standardized and have a narrow dynamic range, which requires dilution of concentrated specimens. We developed and validated an antibody-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for CgA without these limitations. METHODS: CgA was extracted from serum using a mixed-mode anion exchange solid-phase extraction plate, digested with trypsin, and analyzed by LC-MS/MS using well-characterized CgA calibration standards. After validation, the mass spectrometry method was compared with the CISBIO immunoassay using 200 serum specimens previously submitted for CgA analysis. Specimens with discordant results were reanalyzed by high-resolution mass spectrometry- (HRMS) -based methods to assess the contribution of truncated and post-translationally modified forms of CgA. RESULTS: The assay had a linear range of 50 to 50 000 ng/mL, recoveries between 89% and 115%, and intra- and interassay imprecision <10%. LC-MS/MS assay results showed a Pearson's correlation of r = 0.953 with the CISBIO immunoassay, with CgA values being a mean 2- to 4-fold higher. Concordance for CgA between the 2 assays was 80.9% (95% CI 72.8%-89.2%), showing substantial agreement. Truncation and posttranslational modification, including 2 phosphorylation sites that had not been previously observed or predicted to our knowledge, did not appear to contribute directly to discordance between the 2 assays. CONCLUSION: Quantification of CgA by LC-MS/MS provides an analytically sensitive and reproducible alternative to commercially available immunoassays.

The effects of brief high intensity light on ocular growth in chicks developing myopia vary with time of day.

Evidence suggests that the relevant variable in the anti-myopigenic effect of increased time spent outdoors is the increase in light intensity. Because light is the strongest Zeitgeber, it is plausible that the effects of bright light exposure depend on time of day, and may impact circadian rhythms. In these studies, we asked whether the effects on eye growth rates and ocular rhythms of brief daily exposures to bright light differed depending on time of day in eyes developing myopia in response to form deprivation (FD) or negative lens-induced hyperopic defocus (LENS). We also studied the effects of concurrent exposures to brief hyperopic defocus and bright light. Exp. 1: Starting at 12d, chicks wearing monocular diffusers or -10 D lenses were exposed to 3 daily hours (h) of bright light (30K lux) in the morning (FD: n = 12; LENS: n = 7) or evening (FD: n = 21; LENS: n = 7) for a total of 6 exposures. Controls wore diffusers or lenses but weren't exposed to bright light ("not bright" FD: n = 14; LENS: n = 9). Exp. 2: Untreated chicks were exposed to 3 h bright light in the morning (n = 12) or evening (n = 14) for a total of 6 exposures. Controls were not exposed to bright light (n = 11). Exp. 3: Chicks were exposed to 2 h simultaneous monocular hyperopic defocus and bright light in the morning (n = 11), mid-day (n = 7) or evening (n = 8) for 5 exposures. "Not bright" lens-wearing controls were data from published work (Nickla et al., 2017). High frequency A-scan ultrasonography was done at the start and end to measure growth rates. The FD group in Exp. 1 and the morning and evening groups in Exp. 3 were measured at 6-h intervals over the final 24 h to determine parameters for the rhythms in axial length and choroidal thickness. 1. Brief bright light in the evening inhibited eye growth in eyes wearing diffusers or lenses relative to "not bright" controls(interocular differences: FD: 316 vs 468 µm, p = 0.026; LENS: 233 vs 438 µm, p = 0.03); morning bright light had no effect. There was no differential effect of time of day of exposure on the rhythm in axial length; for choroid thickness, "time" accounted for the variance between groups (2-way ANOVA interaction p = 0.027). 2. In binocularly untreated chicks, bright light in the morning had a small but significant growth stimulatory effect relative to evening exposures (803  vs 679 µm/7d; post-hoc p = 0.048). 3. Eyes exposed to simultaneous hyperopic defocus and bright light were significantly more inhibited relative to "not bright" controls for morning exposures (interocular differences: -207 vs 69 µm; p < 0.01). In conclusion, the effects of brief periods of bright light on the growth controller depended on the time of day of exposure and on the contemporaneous state ofocular growth .

Strain-Enhanced Metallic Intermixing in Shape-Controlled Multilayered Core-Shell Nanostructures: Toward Shaped Intermetallics.

Controlling the surface composition of shaped bimetallic nanoparticles could offer precise tunability of geometric and electronic surface structure for new nanocatalysts. To achieve this goal, a platform for studying the intermixing process in a shaped nanoparticle was designed, using multilayered Pd-Ni-Pt core-shell nanocubes as precursors. Under mild conditions, the intermixing between Ni and Pt could be tuned by changing layer thickness and number, triggering intermixing while preserving nanoparticle shape. Intermixing of the two metals is monitored using transmission electron microscopy. The surface structure evolution is characterized using electrochemical methanol oxidation. DFT calculations suggest that the low-temperature mixing is enhanced by shorter diffusion lengths and strain introduced by the layered structure. The platform and insights presented are an advance toward the realization of shape-controlled multimetallic nanoparticles tailored to each potential application.

Effects of time-of-day on inhibition of lens-induced myopia by quinpirole, pirenzepine and atropine in chicks.

Injections of the D2 dopamine receptor agonist quinpirole or the acetylcholine muscarinic receptor antagonists pirenzepine and atropine prevent the development of negative-lens-induced myopia in chicks by inhibiting ocular growth. Because ocular growth is diurnally rhythmic, we hypothesized that the efficacy for inhibition may depend on time of day. Chicks wore monocular -10D lenses for 5 days, starting at 12d of age. The light cycle was 12L/12D. The lens-wearing eye received daily intravitreal injections for 4 days, of 20 µl quinpirole (10 nmol), at the following times: 7:30 EST (lights-on; morning; n = 12), 12:00 (mid-day; n = 13), or 19:30 (evening; n = 17). The same protocol was used for pirenzepine (0.2 µmol) and atropine (18 nmol), at the following times: 8:30 EDT (lights-on; n = 10; n = 18), 14:00 (n = 10; n = 12), or 20:30 (n = 18; n = 16). Saline injections were done in separate groups of birds for all groups as controls, and the data combined (n = 28). Ocular dimensions were measured using A-scan ultrasonography on treatment day 1 at 12:00, and again on day 5 at 12:00; growth rate is defined as the change in axial length over 96 h. For quinpirole and pirenzepine, subsets (n's in Methods) of mid-day and evening groups were measured at 6 h intervals on day 5 (from 12:00 to 12:00) to obtain rhythm parameters for axial length and choroidal thickness; for atropine, only the mid-day group was measured. Refractions were measured on day 5 with a Hartinger's refractometer. For quinpirole and pirenzepine, mid-day injections were more effective at inhibiting ocular growth than evening (Exp-fellow: quinpirole: -68 vs 118 µm/96h; post-hoc Bonferroni p = 0.016; pirenzepine: 79 vs 215 µm/96h; p = 0.046). There were no between-group statistically significant differences for atropine. For quinpirole, the mid-day amplitude of the axial rhythm was smaller than for evening (95 vs 142 µm; p < 0.05), but there were no time-dependent effects on the rhythms for pirenzepine. For atropine, the amplitude of the axial-length rhythm was significantly larger than that for pirenzepine at mid-day. We conclude that there is a phase-dependent efficacy for quinpirole and pirenzepine, with mid-day injections being most effective. There were no consistent time-dependent alterations in rhythm parameters for any of the drugs.

Adamantane/Cucurbituril: A Potential Pretargeted Imaging Strategy in Immuno-PET.

Positron emission tomography (PET) imaging with biological macromolecules greatly expands the possibilities of molecular imaging. There are, however, practical aspects limiting the potential of the approach, including the dosimetric consequences of the slow kinetics of radiolabeled biomacromolecules. Pretargeting strategies have led to impactful improvements in the field but are themselves limited by shortcomings of available bioconjugation methodology. We report our initial findings concerning the suitability of the adamantane/cucurbit[7]uril system for pretargeted immuno-PET imaging and provide proof-of-concept PET/computed tomography imaging experiments to establish the stability and rapid formation of host-guest complexes in vivo. The adamantane/cucurbit[7]uril system itself without antibody conjugation has shown remarkably fast association kinetics and clearance in vivo. We further demonstrate the modulation of biodistribution achievable by cucurbituril complexation with relevance for pharmaceutical formulation as well as the radiosynthetic access to relevant reporter molecules labeled with 11C or 18F. This work, an early proof-of-concept, supports the notion that the adamantane/cucurbit[7]uril system warrants further exploration in pretargeted PET imaging applications.

Brief hyperopic defocus or form deprivation have varying effects on eye growth and ocular rhythms depending on the time-of-day of exposure.

It is generally accepted that myopic defocus is a more potent signal to the emmetropization system than hyperopic defocus: one hour per day of myopic defocus cancels out 11 h of hyperopic defocus. However, we have recently shown that the potency of brief episodes of myopic defocus at inhibiting eye growth depends on the time of day of exposure. We here ask if this will also be true of the responses to brief periods of hyperopic defocus: may integration of the signal depend on time of day? If so, are the rhythms in axial length and choroidal thickness altered? Hyperopic defocus: Birds had one eye exposed to hyperopic defocus by the wearing of -10D lenses for 2 or 6 h at one of 3 times of day for 5 days: Morning (7 am - 9 am: n = 13; 7 am - 1 pm: n = 6), Mid-day (12 pm - 2 pm: n = 20; 10 am - 4 pm: n = 8), or Evening (7 pm - 9 pm: n = 12; 2 pm - 8 pm: n = 11). A separate group wore monocular lenses continually as a control (n = 12). Form deprivation: Birds wore a diffuser over one eye for 2 h at one of 3 times of day for 5 days: Morning (n = 12); Mid-day (n = 19) or Evening (n = 6). For all groups, ocular dimensions were measured using high-frequency A-scan ultrasonography at noon on the first day, under inhalation anesthesia. On day 5, eye dimensions were re-measured at 12 pm, and refractive errors were measured using a Hartinger's refractometer. A subset of birds in the 2-h lens group (morning, n = 8; mid-day, n = 8; evening, n = 6), and the deprivation group (n = 6 per time point), were also measured at 6 pm, 12 am, 6 am and 12 pm on the last day of exposure, to obtain the parameters of the diurnal rhythms in axial length and choroidal thickness. The effects of 2 h of defocus depended on time of day of exposure: it stimulated eye growth when exposure was in the morning and inhibited it when it was at mid-day (change in vitreous chamber, X-C; ANOVA p < 0.0005; 120 µm vs -77 µm/5d, respectively; t-tests: p = 0.001; p = 0.01; post-hoc tests: p = 0.002). For mid-day, experimental eyes were more hyperopic (1.4 D; p < 0.0001). Similar to 2 h defocus, 6 h exposures at mid-day inhibited growth and produced hyperopia (X-C: -167 µm; t-test p = 0.005; RE: 1.8 D; p = 0.03). The effects of 2 h of FD were similar to those of hyperopic defocus in inhibiting growth for mid-day exposures, but FD inhibited growth for the morning exposures as well (Axial length: X-C: Morning: -122 µm; mid-day: -92 µm; ttests p = 0.006 and p = 0.016 respectively). Experimental eyes were more hyperopic (1.8 D; 1.0 D; p < 0.05). The rhythms in axial length were altered for the morning exposures in both conditions. Form deprivation in the morning, which caused inhibition, caused the phases of the two rhythms to shift toward one another (peaks at 6:00 am and 10:45 am for choroid and axial length respectively). Our findings imply that the retinal "integrator", and/or scleral growth regulator exhibit diurnal rhythms. Furthermore, they suggest that reading activities early in the day may be contraindicated in school children at risk of becoming myopic.

A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial agents.

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC50 9.0±0.2µM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Endovascular Reconstruction of Aortoiliac Occlusive Disease with an Aberrant Pelvic Kidney.

Aortoiliac occlusive disease coincident with a congenital ectopic pelvic kidney is a rare occurrence. Traditionally, the treatment has been open aortobifemoral repair with reimplantation of the renal artery. We present a patient with Trans-Atlantic Inter-Society Consensus (TASC) D bilateral aortoiliac occlusive disease, an ectopic pelvic kidney, and prohibitive medical comorbidities. We describe a totally endovascular repair using a chronic total occlusion crossing device, a luminal re-entry device, and balloon-mounted covered stents to revascularize the lower extremities and the ectopic pelvic kidney. We discuss various aspects of this endovascular approach as the incidence of patients with TASC D lesions and prohibitive comorbidities continue to rise.

Public Health System Response to Extreme Weather Events.

CONTEXT: Extreme weather events, unpredictable and often far-reaching, constitute a persistent challenge for public health preparedness. OBJECTIVE: The goal of this research is to inform public health systems improvement through examination of extreme weather events, comparing across cases to identify recurring patterns in event and response characteristics. DESIGN: Structured telephone-based interviews were conducted with representatives from health departments to assess characteristics of recent extreme weather events and agencies' responses. Response activities were assessed using the Centers for Disease Control and Prevention Public Health Emergency Preparedness Capabilities framework. Challenges that are typical of this response environment are reported. SETTING: Forty-five local health departments in 20 US states. RESULTS: Respondents described public health system responses to 45 events involving tornadoes, flooding, wildfires, winter weather, hurricanes, and other storms. Events of similar scale were infrequent for a majority (62%) of the communities involved; disruption to critical infrastructure was universal. Public Health Emergency Preparedness Capabilities considered most essential involved environmental health investigations, mass care and sheltering, surveillance and epidemiology, information sharing, and public information and warning. Unanticipated response activities or operational constraints were common. CONCLUSIONS: We characterize extreme weather events as a "quadruple threat" because (1) direct threats to population health are accompanied by damage to public health protective and community infrastructure, (2) event characteristics often impose novel and pervasive burdens on communities, (3) responses rely on critical infrastructures whose failure both creates new burdens and diminishes response capacity, and (4) their infrequency and scale further compromise response capacity. Given the challenges associated with extreme weather events, we suggest opportunities for organizational learning and preparedness improvements.

Endovascular treatment of an aortic saddle embolism resulting in full neurologic recovery.

Aortic saddle embolism (ASE) and aortic saddle thrombosis are rare and occasionally associated with spinal ischemia and paraplegia. Patients have traditionally been treated with transfemoral balloon thromboembolectomy. In the following case report, we present a patient with suspected ASE and paraplegia, who was successfully treated by an endovascular approach using covered stents. Following intervention, the patient regained full neurologic function. To our knowledge, it is the first time that this type of endovascular treatment for ASE has been applied successfully.

Extra-anatomic endovascular repair of an abdominal aortic aneurysm with a horseshoe kidney supplied by the aneurysmal aorta.

Abdominal aortic aneurysm complicated by a horseshoe kidney (HSK, fused kidney) represents a unique challenge for repair. Renal arteries arising from the aneurysmal aorta can further complicate intervention. Reports exist describing the repair of these complex anatomies using fenestrated endografts, hybrid open repairs (debranching), and open aneurysmorrhaphy with preservation of renal circulation. We describe an extra-anatomic, fully endovascular repair of an abdominal aortic aneurysm with a HSK partially supplied by a renal artery arising from the aneurysm. We successfully applied aortouni-iliac endografting, femorofemoral bypass, and retrograde renal artery perfusion via the contralateral femoral artery to exclude the abdominal aortic aneurysm and preserve circulation to the HSK.

Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition.

Many species of bacteria secrete natural products that inhibit the growth or development of competing species. In turn, competitors may develop or acquire resistance to antagonistic molecules. Few studies have investigated the interplay of these countervailing forces in direct competition between two species. We have used an imaging mass spectrometry (IMS) approach to track metabolites exchanged between Bacillus subtilis and Streptomyces sp. Mg1 cultured together. Surfactin is a cyclic lipopeptide produced by B. subtilis that inhibits the formation of aerial hyphae by streptomycetes. IMS analysis exposed an addition of 18 mass units to surfactin in the agar proximal to Streptomyces sp. Mg1 but not other streptomycetes tested. The spatially resolved change in the mass of surfactin indicated hydrolysis of the molecule. We observed that the aerial growth of Streptomyces sp. Mg1 was resistant to inhibition by surfactin, which suggests that hydrolysis was a mechanism of resistance. To identify possible enzymes from Streptomyces sp. Mg1 with surfactin hydrolase activity, we isolated secreted proteins and identified candidates by mass spectrometry. We purified one candidate enzyme that hydrolyzed surfactin in vitro. We tested the role of this enzyme in surfactin resistance by deleting the corresponding gene from the S. Mg1 genome. We observed that aerial growth by the ΔsfhA mutant strain was now sensitive to surfactin. Our results identify an enzyme that hydrolyzes surfactin and confers resistance to aerial growth inhibition, which demonstrates the effective use of an IMS approach to track natural product modifications during interspecies competition.

Mass spectral molecular networking of living microbial colonies.

Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a "holy grail" in microbiology. This work describes a highly sensitive, broadly applicable, and cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097-1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.

Antiplatelet therapy before, during, and after extremity revascularization.

Peripheral arterial disease (PAD) is well recognized as a marker for systemic atherosclerosis. Platelets play an essential role in all stages of the disease, contributing to both thrombosis and the development of atherosclerosis. Medication regimens to optimize outcomes in both patients who are to undergo revascularization and those who will be managed without interventional therapy must address antiplatelet therapy. Given the common cardiovascular and cerebrovascular comorbidities in patients with PAD, antiplatelet therapy has the potential to decrease thromboembolic events in addition to improving patency after interventions. This clinical update reviews the current literature and recommendations for antiplatelet therapy in patients with PAD.

Estrogen deficiency in the menopause and the role of hormone therapy: integrating the findings of basic science research with clinical trials.

ABSTRACT: Menopause, defined by the cessation of menstrual cycles after 12 months of amenorrhea not due to other causes, is associated with significant hormonal changes, primarily a decrease in estrogen, androgen, and progesterone levels. This review delves into the effects of estrogen deficiency during the perimenopausal transition and postmenopause, integrating the findings of basic science with clinical trials. Here, we first outline the variation in endogenous estrogens before and after menopause, exploring both genomic and nongenomic actions of estrogen and its estrogen receptors throughout the body. Next, we detail the spectrum of menopausal symptoms, from acute vasomotor, urogenital, and psychological issues during perimenopause to chronic reproductive, cardiovascular, neurological, skeletal, dermatologic, immune, and digestive changes postmenopause. Finally, we evaluate the role of hormone therapy in alleviating these symptoms, weighing its benefits against known risks. Publicizing these findings and an accurate representation of the risks and benefits of estrogen replacement to our aging patients is fundamental to improving their care, quality, and even quantity of life.

Similar excitability through different sodium channels and implications for the analgesic efficacy of selective drugs.

Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.

Uniform trehalose nanogels for glucagon stabilization.

Glucagon is a peptide hormone that acts via receptor-mediated signaling predominantly in the liver to raise glucose levels by hepatic glycogen breakdown or conversion of noncarbohydrate, 3 carbon precursors to glucose by gluconeogenesis. Glucagon is administered to reverse severe hypoglycemia, a clinical complication associated with type 1 diabetes. However, due to low stability and solubility at neutral pH, there are limitations in the current formulations of glucagon. Trehalose methacrylate-based nanoparticles were utilized as the stabilizing and solubilizing moiety in the system reported herein. Glucagon was site-selectively modified to contain a cysteine at amino acid number 24 to covalently attach to the methacrylate-based polymer containing pyridyl disulfide side chains. PEG2000 dithiol was employed as the crosslinker to form uniform nanoparticles. Glucagon nanogels were monitored in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 at various temperatures to determine its long-term stability in solution. Glucagon nanogels were stable up to at least 5 months by size uniformity when stored at -20 °C and 4 °C, up to 5 days at 25 °C, and less than 12 hours at 37 °C. When glucagon stability was studied by either HPLC or thioflavin T assays, the glucagon was intact for at least 5 months at -20 °C and 4 °C within the nanoparticles at -20 °C and 4 °C and up to 2 days at 25 °C. Additionally, the glucagon nanogels were studied for toxicity and efficacy using various assays in vitro. The findings indicate that the nanogels were nontoxic to fibroblast cells and nonhemolytic to red blood cells. The glucagon in the nanogels was as active as glucagon alone. These results demonstrate the utility of trehalose nanogels towards a glucagon formulation with improved stability and solubility in aqueous solutions, particularly useful for storage at cold temperatures.

CGG-repeat length threshold for FMR1 RNA pathogenesis in a cellular model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The presence of elevated levels of expanded mRNA found in premutation carriers is believed to be the basis for the pathogenesis in FXTAS, but the exact mechanisms by which the mRNA causes toxicity are not known. In particular, it is not clear whether there is a threshold for a CGG-repeat number below which no cellular dysregulation occurs, or whether toxicity depends on mRNA concentration. We have developed a doxycycline-inducible episomal system that allows us to study separately the effects of CGG-repeat number and mRNA concentration (at fixed CGG-repeat length) in neuroblastoma-derived SK cells. Our findings show that there is a CGG-repeat size threshold for toxicity that lies between 62 and 95 CGG repeats. Interestingly, for repeat sizes of 95 CGG and above, there is a clear negative correlation between mRNA concentration and cell viability. Taken together, our results provide evidence for an RNA-toxicity model with primary dependence on CGG-repeat size and secondary dependence on mRNA concentration, thus formally ruling out any simple titration model that operates in the absence of either protein-binding cooperativity or some form of length-dependent RNA structural transition.