From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

Distinct Inflammatory Phenotypes are associated with subclinical and clinical Cardiovascular disease in People living with HIV.

Despite inflammation being implicated in cardiovascular disease (CVD) in people with HIV (PWH), considerable heterogeneity within populations of PWH exists. Stratifying CVD risk based on inflammatory phenotype could play an important role. Using principal component analyses and unsupervised hierarchical clustering, we examined 38 biomarkers to identify inflammatory phenotypes in two independent cohorts of PWH. We identified three distinct inflammatory clusters present in both cohorts that associated with altered risk of both subclinical CVD (cohort 1) and prevalent clinical CVD (cohort 2) after adjusting for CVD risk factors. These data support precision medicine approaches to enhance CVD risk assessment in PWH.

Affinity Constants of Bovine Serum Albumin for 5 nm Gold Nanoparticles (AuNPs) with ω-Functionalized Thiol Monolayers Determined by Fluorescence Spectroscopy.

A detailed understanding of the binding of serum proteins to small (dcore <10 nm) nanoparticles (NPs) is essential for the mediation of protein corona formation in next generation nanotherapeutics. While a number of studies have investigated the details of protein adsorption on large functionalized NPs, small NPs (with a particle surface area comparable in size to the protein) have not received extensive study. This study determined the affinity constant (Ka) of BSA when binding to three different functionalized 5 nm gold nanoparticles (AuNPs). AuNPs were synthesized using three ω-functionalized thiols (mercaptoethoxy-ethoxy-ethanol (MEEE), mercaptohexanoic acid (MHA), and mercaptopentyltrimethylammonium chloride (MPTMA)), giving rise to particles with three different surface charges. The binding affinity of bovine serum albumin (BSA) to the different AuNP surfaces was investigated using UV-visible absorbance spectroscopy, dynamic light scattering (DLS), and fluorescence quenching titrations. Fluorescence titrations indicated that the affinity of BSA was actually highest for small AuNPs with a negative surface charge (MHA-AuNPs). Interestingly, the positively charged MPTMA-AuNPs showed the lowest Ka for BSA, indicating that electrostatic interactions are likely not the primary driving force in binding of BSA to these small AuNPs. Ka values at 25 °C for MHA, MEEE, and MPTMA-AuNPs were 5.2 ± 0.2 × 107, 3.7 ± 0.2 × 107, and 3.3 ± 0.16 × 107 M-1 in water, respectively. Fluorescence quenching titrations performed in 100 mM NaCl resulted in lower Ka values for the charged AuNPs, while the Ka value for the MEEE-AuNPs remained unchanged. Measurement of the hydrodynamic diameter (Dh) by dynamic light scattering (DLS) suggests that adsorption of 1-2 BSA molecules is sufficient to saturate the AuNP surface. DLS and negative-stain TEM images indicate that, despite the lower observed Ka values, the binding of MPTMA-AuNPs to BSA likely induces significant protein misfolding and may lead to extensive BSA aggregation at specific BSA:AuNP molar ratios.

Developing a Computer Vision Model to Automate Quantitative Measurement of Hip-Knee-Ankle Angle in Total Hip and Knee Arthroplasty Patients.

BACKGROUND: Increasing deformity of the lower extremities, as measured by the hip-knee-ankle angle (HKAA), is associated with poor patient outcomes after total hip and knee arthroplasty (THA, TKA). Automated calculation of HKAA is imperative to reduce the burden on orthopaedic surgeons. We proposed a detection-based deep learning (DL) model to calculate HKAA in THA and TKA patients and assessed the agreement between DL-derived HKAAs and manual measurement. METHODS: We retrospectively identified 1,379 long-leg radiographs (LLRs) from patients scheduled for THA or TKA within an academic medical center. There were 1,221 LLRs used to develop the model (randomly split into 70% training, 20% validation, and 10% held-out test sets); 158 LLRs were considered "difficult," as the femoral head was difficult to distinguish from surrounding tissue. There were 2 raters who annotated the HKAA of both lower extremities, and inter-rater reliability was calculated to compare the DL-derived HKAAs with manual measurement within the test set. RESULTS: The DL model achieved a mean average precision of 0.985 on the test set. The average HKAA of the operative leg was 173.05 ± 4.54°; the nonoperative leg was 175.55 ± 3.56°. The inter-rater reliability between manual and DL-derived HKAA measurements on the operative leg and nonoperative leg indicated excellent reliability (intraclass correlation (2,k) = 0.987 [0.96, 0.99], intraclass correlation (2, k) = 0.987 [0.98, 0.99, respectively]). The standard error of measurement for the DL-derived HKAA for the operative and nonoperative legs was 0.515° and 0.403°, respectively. CONCLUSIONS: A detection-based DL algorithm can calculate the HKAA in LLRs and is comparable to that calculated by manual measurement. The algorithm can detect the bilateral femoral head, knee, and ankle joints with high precision, even in patients where the femoral head is difficult to visualize.

Validation of the bag-mediated filtration system for environmental surveillance of poliovirus in Nairobi, Kenya.

AIMS: This study compared the bag-mediated filtration system (BMFS) and standard WHO two-phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin-like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. METHODS AND RESULTS: Environmental samples were collected in Nairobi, Kenya (Sept 2015-Feb 2017), concentrated via BMFS and two-phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two-phase samples (P < 0·05). In BMFS samples, SL PV detection did not significantly differ with volume filtered, filtration time or filter shipment time (P > 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two-phase samples (P < 1 × 10-5 ). CONCLUSIONS: Poliovirus was detected at higher rates with the BMFS, a method that includes a secondary concentration step, than using the standard WHO two-phase method. SL2 disappearance from the environment was commensurate with OPV2 withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: The BMFS offers comparable or improved PV detection under the conditions in this study, relative to the two-phase method.

Rhenium-Based Complexes and in Vivo Testing: A Brief History.

The success of metal-based anticancer therapeutics in the treatment of cancer is best exemplified by cisplatin. Currently used in 32/78 cancer regimens, metal-based drugs have a clear role in cancer therapy. Despite this, metal-based anticancer therapeutics are not without drawbacks, with issues such as toxic side effects and the development of resistance mechanisms. This has led to investigations of other metal-based drug candidates such as auranofin, a gold-based drug candidate as well as ruthenium-based candidates, NAMI-A, NKP-1339 and TLD-1433. All are currently undergoing clinical trials. Another class of complexes under study are rhenium-based; such complexes have undergone extensive in vitro testing but only nine have been reported to display antitumour in vivo activity, which is a necessary step before entering clinical trials. This review will document, chronologically, the rhenium-based drug candidates that have undergone in vivo testing and the outlook for such complexes.

Cytotoxicity, cellular localization and photophysical properties of Re(I) tricarbonyl complexes bound to cysteine and its derivatives.

The potential chemotherapeutic properties coupled to photochemical transitions make the family of fac-[Re(CO)3(N,N)X]0/+ (N,N = a bidentate diimine such as 2,2'-bipyridine (bpy); X = halide, H2O, pyridine derivatives, PR3, etc.) complexes of special interest. We have investigated reactions of the aqua complex fac-[Re(CO)3(bpy)(H2O)](CF3SO3) (1) with potential anticancer activity with the amino acid L-cysteine (H2Cys), and its derivative N-acetyl-L-cysteine (H2NAC), as well as the tripeptide glutathione (H3A), under physiological conditions (pH 7.4, 37 °C), to model the interaction of 1 with thiol-containing proteins and enzymes, and the impact of such coordination on its photophysical properties and cytotoxicity. We report the syntheses and characterization of fac-[Re(CO)3(bpy)(HCys)]·0.5H2O (2), Na(fac-[Re(CO)3(bpy)(NAC)]) (3), and Na(fac-[Re(CO)3(bpy)(HA)])·H2O (4) using extended X-ray absorption spectroscopy, IR and NMR spectroscopy, electrospray ionization spectrometry, as well as the crystal structure of {fac-[Re(CO)3(bpy)(HCys)]}4·9H2O (2 + 1.75 H2O). The emission spectrum of 1 displays a variance in Stokes shift upon coordination of L-cysteine and N-acetyl-L-cysteine. Laser excitation at λ = 355 nm of methanol solutions of 1-3 was followed by measuring their ability to produce singlet oxygen (1O2) using direct detection methods. The cytotoxicity of 1 and its cysteine-bound complex 2 was assessed using the MDA-MB-231 breast cancer cell line, showing that the replacement of the aqua ligand on 1 with L-cysteine significantly reduced the cytotoxicity of the Re(I) tricarbonyl complex. Probing the cellular localization of 1 and 2 using X-ray fluorescence microscopy revealed an accumulation of 1 in the nuclear and/or perinuclear region, whereas the accumulation of 2 was considerably reduced, potentially explaining its reduced cytotoxicity. Replacing the aqua ligand with cysteine in the antitumor active fac-[Re(CO)3(bpy)(H2O)](CF3SO3) complex significantly reduced its cellular accumulation and cytotoxicity against the MDA-MB-213 breast cancer cell line, shifted its maximum emission to considerably higher energies, and decreased its fluorescence quantum yield.

A computationally engineered RAS rheostat reveals RAS-ERK signaling dynamics.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors.

Inflammation, immunity, and amyotrophic lateral sclerosis: II. immune-modulating therapies.

With the emerging popularity of immune-modulatory therapies to treat human diseases there is a need to step back from hypotheses aimed at assessing a condition in a single-system context and instead take into account the disease pathology as a whole. In complex diseases, such as amyotrophic lateral sclerosis (ALS), the use of these therapies to treat patients has been largely unsuccessful and likely premature given our lack of understanding of how the immune system influences disease progression and initiation. In addition, we still have an incomplete understanding of the role of these responses in our model systems and how this may translate clinically to human patients. In this review we discuss preclinical evidence and clinical trial results for a selection of recently conducted studies in ALS. We provide evidence-based reasoning for the failure of these trials and offer suggestions to improve the design of future investigations. Muscle Nerve 59:23-33, 2019.

Inflammation, Immunity, and amyotrophic lateral sclerosis: I. Etiology and pathology.

Amyotrophic lateral sclerosis (ALS) is a severely debilitating disease characterized by progressive degeneration of motor neurons. Charcot first described ALS in 18691 ; however, its pathogenesis remains unknown, and effective treatments remain elusive. It is apparent that new paradigms must be investigated to understand the effectors of ALS, including inflammation, immune responses, and the body's response to stress and injury. Herein we discuss the potential role of the immune system in ALS pathogenesis and critically review evidence from patient and animal studies. Although immune system components may indeed play a role in ALS pathogenesis, studies implicating immune cells, antibodies, and cytokines in early disease pathology are limited. We propose more focused studies that examine the role of the immune system together with characterized pathogenesis to determine when, where, and if immune and inflammatory processes are critical to disease progression, and thus worthy targets of intervention. Muscle Nerve 59:10-22, 2019.

The first report of cerebral, pulmonary and gastrointestinal angiostrongyliasis in the water rat Hydromys chrysogaster from Queensland, Australia.

We describe the first case of angiostrongyliasis in a water rat, Hydromys chrysogaster, a large rodent adapted to aquatic life, which is endemic to Australia, New Guinea and adjacent islands.

Ageing impairs parasite-specific antibody responses in cystic echinococcosis.

Humoral immunity wanes during healthy ageing, increasing susceptibility to infections in the elderly. In this sense, information about parasite infections and human immunosenescence is scarce. Cystic echinococcosis (CE) is an infectious disease caused by the larval stage of the cestode parasite Echinococcus granulosus, whose prevalence in humans shows an increase with host age. Susceptibility to human CE has been associated with humoral immunity to some extent, and, therefore, we have here analysed the influence of host age on the serological profile of young, middle-aged and aged patients. Our results highlighted the detrimental influence of ageing on the intensity and quality of the antiparasite antibody response. Remarkable differences in serological profiles between young and aged individuals were observed. In this sense, through Principal Components Analysis, we identified aged patients as those exhibiting overall less intense antibody responses, mainly in isotypes/subclasses supposed to exert efficient antiparasite activities (eg IgE and IgG1). Thus, these humoral defects could at least partially explain the reported increase in CE prevalence among older individuals, as a weaker immune response in the elderly might facilitate the establishment and maintenance of the parasite infection. Finally, a possible association between age-dependent susceptibility to CE and host immunosenescence is discussed.

Antibody profiling in ultrasound normal individuals with positive serology for cystic echinococcosis.

Cystic echinococcosis is a zoonotic disease caused by the cestode parasite Echinococcus granulosus. In endemic regions, seropositive individuals to E. granulosus usually and markedly outnumber image-confirmed cases of cystic echinococcosis, suggesting that some parasite challenges derive in unsuccessful infection establishments. However, it is still unknown whether such parasite-specific antibodies in healthy individuals might play a role in resistance/susceptibility to the infection. Therefore, we have here analysed the profile of antibodies recognizing E. granulosus antigens in seropositive but ultrasound normal individuals, as well as in surgery-confirmed patients and healthy donors. Our results showed that ultrasound normal individuals exhibited low avidity IgG antibodies, as well as low levels of parasite-specific IgG1 and IgG4 antibodies. In addition, they displayed significant levels of specific IgE, and thus, they revealed a uniquely high IgE:IgG4 ratio. Moreover, high levels of parasite-specific IgM were detected in such individuals, which showed characteristics of natural cross-reacting antibodies. Therefore, our results indicate that ultrasound normal individuals but seropositive for E. granulosus antigens exhibit a distinctive antibody profile. In this regard, possible associations between their antiparasite antibodies and potential resistance mechanisms to cystic echinococcosis are discussed.

Can Marginal Rates of Substitution Be Inferred From Happiness Data? Evidence from Residency Choices.

We survey 561 students from U.S. medical schools shortly after they submit choice rankings over residencies to the National Resident Matching Program. We elicit (a) these choice rankings, (b) anticipated subjective well-being (SWB) rankings, and (c) expected features of the residencies (such as prestige). We find substantial differences between choice and anticipated-SWB rankings in the implied tradeoffs between residency features. In our data, evaluative SWB measures (life satisfaction and Cantril's ladder) imply tradeoffs closer to choice than does affective happiness (even time-integrated), and as close as do multi-measure SWB indices. We discuss implications for using SWB data in applied work.

Beyond Happiness and Satisfaction: Toward Well-Being Indices Based on Stated Preference.

This paper proposes foundations and a methodology for survey-based tracking of well-being. First, we develop a theory in which utility depends on "fundamental aspects" of well-being, measurable with surveys. Second, drawing from psychologists, philosophers, and economists, we compile a comprehensive list of such aspects. Third, we demonstrate our proposed method for estimating the aspects' relative marginal utilities-a necessary input for constructing an individual-level well-being index-by asking ~4,600 U.S. survey respondents to state their preference between pairs of aspect bundles. We estimate high relative marginal utilities for aspects related to family, health, security, values, freedom, happiness, and life satisfaction.

Integration of the Client Self-Care Commitment Model in a dental hygiene curriculum.

OBJECTIVE: This exploratory study investigated whether integration of the Client Self-Care Commitment Model (CSCCM) Instructional Module in a dental hygiene curriculum, as an additional educational experience, would further enhance students' client-centred knowledge, values and actions. METHODS: Subjects (n = 26) were second-year students enrolled in a BS entry-level dental hygiene programme with random assignments to two groups. The experimental group participated in a 2-h didactic session, an 8-h preclinical session, an 8-h clinical session and a 1-h question and answer period. An online pretest-post-test survey administered at three time intervals (baseline, 3 and 6 weeks) was used to measure differences between the groups on three subscales (knowledge, values and actions). RESULTS: Cronbach's α for each subscale across time was above 0.90. A repeated-measures anova determined that there were no statistically significant interactions between Time and Group (experimental or control group) for the knowledge and values variables; however, there was a significant interaction between Time (P = 0.003) and Group (P = 0.033) for the actions variable. A content analysis of participants' responses to three open-ended questions reflected both positive and negative comments and revealed that students' primary barrier to implementing the model in client care was lack of time. CONCLUSIONS: Significant differences in the actions variable between the groups suggested that implementation of the CSCCM Instructional Module enhanced students' client-centred actions during client care.

The Effect of Uncertainty on Optimal Control Models in the Neighbourhood of a Steady State.

For both discrete and continuous time this paper derives the Taylor approximation to the effect of uncertainty (in the simple sense of risk, not Knightian uncertainty) on expected utility and optimal behaviour in stochastic control models when the uncertainty is small enough that one can focus on only the first term that involves uncertainty. There is a close and illuminating relationship between the discrete-time and continuous-time results. The analysis makes it possible to spell out a tight connection between the behaviour of a dynamic stochastic general equilibrium model and the corresponding perfect foresight model. However, the quantitative analytics of the stochastic model local to a certainty model calls for a more thorough investigation of the nearby certainty model than is typically undertaken.

Effects of Ball Size and Weight on Throwing Kinematics and Kinetics in Youth Baseball Athletes.

BACKGROUND: In baseball, youth athletes play on smaller fields with shorter distances between bases, shorter pitching distances, and smaller mounds. Despite this, youth athletes use baseballs weighing the same amount as those used at the professional level, possibly predisposing youth baseball players to injuries. PURPOSE: (1) To determine the effects of throwing a smaller, lighter, and both smaller and lighter baseball on throwing arm stress in youth athletes and (2) to also investigate how changing the ball size and weight would affect elbow varus torque, shoulder distraction force, and throwing arm internal rotation velocity during the throwing motion. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: This cross-sectional cohort study analyzed the kinematics and kinetics of 38 youth baseball players (mean age, 8.3 ± 0.8 years) throwing a baseball modified in size and weight. Three-dimensional motion data were collected using a retroreflective marker set and a 12-camera motion analysis system. Full-body kinematics and kinetics were calculated using commercial software. Participants threw 5 different types of baseballs 3 times each, in random order, with full effort from a pitching mound to a target 14 m away. The balls used were a 5-oz regular baseball, 5-oz (0.142-kg) baseball with a 5% reduced circumference, 4-oz (0.113-kg) baseball, 4-oz baseball with a 5% reduced circumference, and 3-oz (0.085-kg) baseball. Analysis of variance was used to determine statistical differences in elbow varus torque, shoulder distraction force, and throwing arm internal rotation velocity among baseball types. The Tukey post hoc test was used to further investigate differences between the ball groups, considering P < .05 to be significant. RESULTS: Analysis of variance detected a significant difference in elbow varus torque among ball groups (P = .024). The Tukey post hoc test revealed a moderate difference in elbow varus torque between the 5-oz baseball (4.73 ± 1.06 percentage body weight × height [%BW × H]) and 3-oz baseball (4.06 ± 0.83 %BW × H) (P = .017; d = 0.677 [95% CI, 0.08-1.27]). No significant differences were found in shoulder distraction force or throwing arm internal rotation velocity among ball groups. CONCLUSION: Compared with a 5-oz baseball, throwing a 3-oz baseball resulted in decreased elbow varus torque with a moderate effect size.

Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.