Broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

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 (LLR) 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 two 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 non-operative leg was 175.55 +/- 3.56°. The inter-rater reliability between manual and DL-derived HKAA measurements on the operative leg and non-operative leg indicated excellent reliability (Intraclass Correlation (ICC) (2,k) = 0.987 [0.96, 0.99], ICC (2,k) = 0.987 [0.98, 0.99, respectively]). The standard error of measurement for the DL-derived HKAA for the operative and non-operative legs was 0.515° and 0.403°, respectively. CONCLUSION: 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.

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.

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.

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.

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.

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. 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 phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the 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, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands.

Blueprinting expandable nanomaterials with standardized protein building 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 due to the irregular shapes of protein structures 1 . 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 been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

Novel Macrocyclic Antagonists of the CGRP Receptor Part 2: Stereochemical Inversion Induces an Unprecedented Binding Mode.

The diastereomeric macrocyclic calcitonin gene-related peptide (CGRP) antagonists HTL0029881 (3) and HTL0029882 (4), in which the stereochemistry of a spiro center is reversed, surprisingly demonstrate comparable potency. X-ray crystallographic characterization demonstrates that 3 binds to the CGRP receptor in a precedented manner but that 4 binds in an unprecedented, unexpected, and radically different manner. The observation of this phenomenon is noteworthy and may open novel avenues for CGRP receptor antagonist design.

Priming enzymes from the pikromycin synthase reveal how assembly-line ketosynthases catalyze carbon-carbon chemistry.

The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KSQ, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KSQ and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KSQ and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KSQ active site, while an AT-swapped version of Pik127 demonstrated that KSQ can also decarboxylate malonyl groups. Mechanisms for how KSQ and KS domains catalyze carbon-carbon chemistry are proposed.

An introduction to gender-affirming hormone therapy for transgender and gender-nonbinary patients.

ABSTRACT: Transgender and gender-nonbinary patients may present to primary care providers seeking gender-affirming hormone therapy. Patients who meet criteria for diagnosis of gender incongruence may start or continue hormone therapy after providing informed consent. Prescribing and monitoring of masculinizing and feminizing hormone therapy can be managed in primary care settings.

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.

Acetylcholine prioritises direct synaptic inputs from entorhinal cortex to CA1 by differential modulation of feedforward inhibitory circuits.

Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.

The effect of sodium thiosulfate on cytotoxicity of a diimine Re(I) tricarbonyl complex.

Recently, diimine Re(i) tricarbonyl complexes have attracted great interest due to their promising cytotoxic effects. Here, we compare the cytotoxicity and cellular uptake of two Re(i) compounds fac-[(Re(CO)3(bpy)(H2O)](CF3SO3) (1) and Na(fac-[(Re(CO)3(bpy)(S2O3)])·H2O (bpy = 2,2'-bipyridine) (2). The Re-thiosulfate complex in 2 was characterized in two solvated crystal structures {Na(fac-[Re(CO)3(bpy)(S2O3)])·1.75H2O·C2H5OH}4 (2 + 0.75H2O + C2H5OH)4 and (fac-[Re(CO)3(bpy)(H2O)]) (fac-[Re(CO)3(bpy)(S2O3)])·4H2O (3). The cytotoxicity of 1 and 2 was tested in the MDA-MB-231 breast cancer cell line and compared with that of cisplatin. The cellular localization of the Re(i) complexes was investigated using synchrotron-based X-ray fluorescence microscopy (XFM). The results show that replacement of the aqua ligand with thiosulfate renders the complex less toxic most likely by distrupting its cellular entry. Therefore, thiosulfate could potentially have a similar chemoprotective effect against diimine fac-Re(CO)3 complexes as it has against cisplatin.

Zinc stable isotope analysis reveals Zn dyshomeostasis in benign tumours, breast cancer, and adjacent histologically normal tissue.

The disruption of Zn homeostasis has been linked with breast cancer development and progression. To enhance our understanding of changes in Zn homeostasis both inside and around the tumour microenvironment, Zn concentrations and isotopic compositions (δ66Zn) were determined in benign (BT) and malignant (MT) tumours, healthy tissue from reduction mammoplasty (HT), and histologically normal tissue adjacent to benign (NAT(BT)) and malignant tumours (NAT(MT)). Mean Zn concentrations in NAT(BT) are 5.5 µg g-1 greater than in NAT(MT) (p = 0.00056) and 5.1 µg g-1 greater than in HT (p = 0.0026). Zinc concentrations in MT are 12.9 µg g-1 greater than in HT (p = 0.00012) and 13.3 µg g-1 greater than in NAT(MT) (p < 0.0001), whereas δ66Zn is 0.17‰ lower in MT than HT (p = 0.017). Benign tumour Zn concentrations are also elevated compared to HT (p = 0.00013), but are not significantly elevated compared to NAT(BT) (p = 0.32). The δ66Zn of BT is 0.15‰ lower than in NAT(BT) (p = 0.045). The similar light δ66Zn of BT and MT compared to HT and NAT may be related to the isotopic compensation of increased metallothionein (64Zn-rich) expression by activated matrix metalloproteinase (66Zn-rich) in MT, and indicates a resultant 66Zn-rich reservoir may exist in patients with breast tumours. Zinc isotopic compositions thus show promise as a potential diagnostic tool for the detection of breast tumours. The revealed differences of Zn accumulation in healthy and tumour-adjacent tissues require additional investigation.

Zinc stable isotopes in urine as diagnostic for cancer of secretory organs.

Breast, prostate, and pancreatic cancers alter the zinc (Zn) metabolism. Combined analyses of urinary Zn concentrations [Zn] and Zn stable isotope compositions (δ66Zn) may provide a non-invasive approach for tracing malignancy-induced Zn dyshomeostasis. In this study, we measured [Zn] and δ66Zn in urine from prostate (n = 22), breast (n = 16), and from women with benign breast disease (n = 14) and compared those with age-matched healthy controls (22-49 years or 50+ years) and published data for pancreatic cancer (n = 17). Our results show that cancer-induced changes are reflected in higher urinary [Zn] and lower urinary δ66Zn for pancreatic and prostate cancer and benign breast disease when compared with healthy controls. For prostate cancer, the progression of low [Zn] and high δ66Zn for patients of low-risk disease toward high [Zn] and low δ66Zn for the higher risk patients demonstrates that [Zn] and δ66Zn in urine could serve as a reliable prognostic tool. Urinary excretion of isotopically light Zn by patients with prostatic and pancreatic cancer is probably the result of increased reactive oxygen species in cancerous cells, which limits the scavenging of hydroxyl radicals and thus facilitates the oxidation of metalloproteins with sulfur-rich ligands. Urine from breast cancer patients shows undistinguishable δ66Zn to healthy controls, implying that the expression of metalloproteins with sulfur-rich ligands is stronger in breast cancer tissues. In conclusion, urinary δ66Zn may provide a non-invasive diagnostic tool for pancreatic cancer and support disease prognosis for prostate cancer. These findings should translate to comprehensive transverse and longitudinal cohort studies in future.

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.

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.

Generalized tonic-clonic seizures with post-ictal atrial fibrillation.

Convulsive seizures are known to cause severe cardiopulmonary changes and increased autonomic activity. Limited reports describe peri-ictal cardiac arrhythmias such as atrial fibrillation (AF) with generalized tonic-clonic seizures (GTCS). We present a unique case of a healthy 23-year-old male patient with new onset prolonged AF in the setting of new onset seizures, occurring on three independent occasions. Over two years, our patient had multiple hospitalizations for seizures with an electrocardiogram (ECG) diagnosis of AF made on three different occasions, occurring during his post-ictal state (all within 30 min of seizure onset). These seizures were never captured by electroencephalography (EEG) or witnessed by the medical staff, but were reported by family and/or reviewed on video provided by them. After his first GTCS, his AF persisted and was medically cardioverted. Two additional instances of AF after witnessed GTCS have been captured. After his second unprovoked seizure, an anti-seizure drug (ASD) was prescribed. A multi-disciplinary approach may be adopted to address comorbidities associated with seizures. Aggressive evaluation and treatment should be employed for newly diagnosed and refractory seizure patients associated with arrhythmias, in our case AF. Peri-ictal arrhythmias may be considered a potential marker for increased sudden unexpected death in epilepsy (SUDEP) risk.