Early Oligocene kelp holdfasts and stepwise evolution of the kelp ecosystem in the North Pacific.

Kelp forests are highly productive and economically important ecosystems worldwide, especially in the North Pacific Ocean. However, current hypotheses for their evolutionary origins are reliant on a scant fossil record. Here, we report fossil hapteral kelp holdfasts from western Washington State, USA, indicating that kelp has existed in the northeastern Pacific Ocean since the earliest Oligocene. This is consistent with the proposed North Pacific origin of kelp associated with global cooling around the Eocene-Oligocene transition. These fossils also support the hypotheses that a hapteral holdfast, rather than a discoid holdfast, is the ancestral state in complex kelps and suggest that early kelps likely had a flexible rather than a stiff stipe. Early kelps were possibly grazed upon by mammals like desmostylians, but fossil evidence of the complex ecological interactions known from extant kelp forests is lacking. The fossil record further indicates that the present-day, multi-story kelp forest had developed at latest after the mid-Miocene climate optimum. In summary, the fossils signify a stepwise evolution of the kelp ecosystem in the North Pacific, likely enabled by changes in the ocean-climate system.

[11C]Paraoxon: Radiosynthesis, Biodistribution and In Vivo Positron Emission Tomography Imaging in Rat.

Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([11C]POX) and profiling in live rats is reported. Naïve rats intravenously injected with [11C]POX showed a rapid decrease in parent tracer to ∼1%, with an increase in radiolabeled serum proteins to 87% and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 minutes, reflecting covalent modification of proteins by [11C]POX. Ex vivo biodistribution and imaging profiles in naïve rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection and persisted over the 60-minute experiment. This showed for the first time that even low POX exposures (∼200 ng tracer) can rapidly enter brain. Rats given an LD50 dose of nonradioactive paraoxon at the LD50 20 or 60 minutes prior to [11C]POX tracer revealed that protein pools were blocked. Blood radioactivity at 20 minutes was markedly lower than naïve levels due to rapid protein modification by nonradioactive POX; however, by 60 minutes the blood radioactivity returned to near naïve levels. Live rat tissue imaging-derived radioactivity values were 10%-37% of naïve levels in nonradioactive POX pretreated rats at 20 minutes, but by 60 minutes the area under the curve (AUC) values had recovered to 25%-80% of naïve. The live rat imaging supported blockade by nonradioactive POX pretreatment at 20 minutes and recovery of proteins by 60 minutes. SIGNIFICANCE STATEMENT: Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX, etc. To study the distribution and penetration of POX into the central nervous system (CNS) and other tissues, a positron emission tomography (PET) tracer analog, carbon-11-labeled paraoxon ([11C]POX), was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.

Paediatric perspectives in the diagnosis of polyuria-polydipsia syndrome.

The elucidation of the underlying cause of polyuria-polydipsia syndrome (PPS) is a challenging-especially in the differentiation of partial defects of arginine vasopressin (AVP) secretion or action from primary polydipsia. The water deprivation test has been utilized for many decades, and its application in the paediatric population has been applied using parameters predominantly established in adult cohorts. In more recent times, the development of automated commercial assays for copeptin, a surrogate marker for AVP, has represented a significant advancement in the diagnostic approach to PPS. Measurement of copeptin concentrations has major advantages and has essentially superseded measurement of AVP in diagnostic protocols for PPS. Additionally, stimulated-copeptin protocols utilizing hypertonic saline infusion, arginine, and glucagon have been investigated, and are promising. However, further studies are required in the population-incorporating the differences in physiological regulation of water homeostasis, and safety requirements-before there is widespread adoption into clinical practice.

Autoregulation of insulin receptor signaling through MFGE8 and the αvß5 integrin.

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvß5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/ß5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvß5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/ß5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvß5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Evaluating Radioactive Analogs of Doxorubicin to Quantify ChemoFilter Binding and Whole-Body Positron Emission Tomography/Magnetic Resonance Imaging for Drug Biodistribution.

PURPOSE: To evaluate radiolabeled doxorubicin (Dox) analogs as tracers of baseline Dox biodistribution in vivo during hepatic intra-arterial chemotherapy and to assess the efficacy of ChemoFilter devices to bind Dox in vitro. MATERIALS AND METHODS: In an in vitro static experiment, [fluorine-18]N-succinimidyl 4-fluorobenzoate ([18F]SFB) and [fluorine-18]fluorobenzoyl-doxorubicin ([18F]FB-Dox) were added to a beaker containing a filter material (Dowex cation exchange resin, single-stranded DNA (ssDNA) resin, or sulfonated polymer coated mesh). In an in vitro flow model, [18F]FB-Dox was added into a Dox solution in phosphate-buffered saline, and the solution flowed via a syringe column containing the filter materials. In an in vitro flow experiment, using micro-positron emission tomography (PET), images were taken as [18F]SFB and [18F]FB-Dox moved through a phantom. For in vivo biodistribution testing, a catheter was placed into the common hepatic artery of a swine, and [18F]FB-Dox was infused over 30 seconds. A 10-minute dynamic image and three 20-minute static images were acquired using 3T PET/MR imaging. RESULTS: In the in vitro static experiment, [18F]FB-Dox demonstrated 76.7%, 88.0%, and 52.4% binding to the Dowex resin, ssDNA resin, and coated mesh, respectively. In the in vitro flow model, the first-pass binding of [18F]FB-Dox to the Dowex resin, ssDNA resin, and coated mesh was 76.7%, 74.2%, and 76.2%, respectively, and the total bound fraction was 80.9%, 84.6%, and 79.9%, respectively. In the in vitro flow experiment using micro-PET, the phantom demonstrated a greater amount of [18F]FB-Dox bound to both filter cartridges than of the control [18F]SFB. In in vivo biodistribution testing, the first 10 minutes depicted [18F]FB-Dox moving through the right upper quadrant of the abdomen. A region-of-interest analysis showed that the relative amount increased by 2.97 times in the gallbladder and 1.08 times in the kidney. The amount decreased by 0.74 times in the brain and 0.57 times in the heart. CONCLUSIONS: [18F]FB-Dox can be used to assess Dox binding to ChemoFilters as well as in vivo biodistribution. This sets the stage for the evaluation of ChemoFilter effectiveness in reducing systemic toxicity from intra-arterial chemotherapy.

Fifty years of newborn screening for congenital hypothyroidism: current status in Australasia and the case for harmonisation.

OBJECTIVES: Since its implementation 50 years ago in Quebec, Canada, newborn screening for congenital hypothyroidism has become one of the most successful public health measures worldwide. Screening programmes across Australia and New Zealand are characterised by significant commonalities in screening algorithms, and a high degree of regional cooperation in harmonisation efforts. We aimed to conduct a comprehensive survey of current performance and practices related to the total testing process for congenital hypothyroidism screening and provide recommendations for harmonisation priorities within our region. METHODS: A survey was conducted involving the six newborn screening laboratories which provide complete geographic coverage across Australasia. Approximately 360,000 newborns are screened annually. Survey questions incorporated pre-analytical, analytical, and post-analytical aspects of the screening programmes and an extensive 5-year (2016-2020) retrospective analysis of individual programme performance data. Responses from individual screening programmes were collated. RESULTS: The uptake of newborn screening was over 98% for the six major jurisdictions. All programmes have adopted a single-tier thyroid stimulating hormone (TSH) strategy using the Perkin Elmer GSP instrument. Significant similarities exist between programmes for recommended age of collection and recollection protocols for low birthweight newborns. The process for the determination of TSH cutoffs varies between programmes. TSH lower cut-offs for borderline-positive and positive notifications between 12-15 and 12-25 mIU/L blood, respectively. Recall rates vary between 0.08 and 0.20%. The case definition for congenital hypothyroidism generally includes biochemical and radiological parameters in addition to the commencement of thyroxine. All programmes reported collecting biochemical and clinical data on infants with positive screening tests, and positive predictive values vary between 23.6 and 77.3%. Variation in reported incidence (1:1,300-2,000) cannot be entirely explained by cutoff or recall rate (although one programme reporting fewer cases includes only permanent disease). CONCLUSIONS: Despite similarities between newborn screening algorithms for congenital hypothyroidism across Australia and New Zealand, differences in reported programme performance provide the basis for further harmonisation. Surveillance of a large population offers the potential for the ongoing development of evidence-based screening guidelines.

Idealness of k-wise intersecting families.

A clutter is k-wise intersecting if every k members have a common element, yet no element belongs to all members. We conjecture that, for some integer k ≥ 4 , every k-wise intersecting clutter is non-ideal. As evidence for our conjecture, we prove it for k = 4 for the class of binary clutters. Two key ingredients for our proof are Jaeger's 8-flow theorem for graphs, and Seymour's characterization of the binary matroids with the sums of circuits property. As further evidence for our conjecture, we also note that it follows from an unpublished conjecture of Seymour from 1975. We also discuss connections to the chromatic number of a clutter, projective geometries over the two-element field, uniform cycle covers in graphs, and quarter-integral packings of value two in ideal clutters.

Biological Distribution and Metabolic Profiles of Carbon-11 and Fluorine-18 Tracers of VX- and Sarin-Analogs in Sprague-Dawley Rats.

Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [18F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.

Strengthening convex relaxations of 0/1-sets using Boolean formulas.

In convex integer programming, various procedures have been developed to strengthen convex relaxations of sets of integer points. On the one hand, there exist several general-purpose methods that strengthen relaxations without specific knowledge of the set S of feasible integer points, such as popular linear programming or semi-definite programming hierarchies. On the other hand, various methods have been designed for obtaining strengthened relaxations for very specific sets S that arise in combinatorial optimization. We propose a new efficient method that interpolates between these two approaches. Our procedure strengthens any convex set containing a set S ⊆ { 0 , 1 } n by exploiting certain additional information about S. Namely, the required extra information will be in the form of a Boolean formula ϕ defining the target set S. The new relaxation is obtained by "feeding" the convex set into the formula ϕ . We analyze various aspects regarding the strength of our procedure. As one application, interpreting an iterated application of our procedure as a hierarchy, our findings simplify, improve, and extend previous results by Bienstock and Zuckerberg on covering problems.

Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Interstitial cells of Cajal, which express the calcium-activated chloride channel transmembrane member 16A (TMEM16A), are an important determinant of gastrointestinal (GI) motility. We previously identified the acylaminocycloalkylthiophene class of TMEM16A inhibitors, which, following medicinal chemistry, gave analog 2-bromodifluoroacetylamino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide (TMinh-23) with 30 nM half-maximal inhibitory concentration. Here, we tested the efficacy of TMinh-23 for inhibition of GI motility in mice. In isolated murine gastric antrum, TMinh-23 strongly inhibited spontaneous and carbachol-stimulated rhythmic contractions. Pharmacokinetic analysis showed predicted therapeutic concentrations of TMinh-23 for at least 4 h following a single oral or intraperitoneal dose at 10 mg/kg. Gastric emptying, as assessed following an oral bolus of phenol red or independently by [99mTc]-diethylenetriamine pentaacetic acid scintigraphy, was reduced by TMinh-23 by ∼60% at 20 min. Interestingly, there was little effect of TMinh-23 on baseline whole-gut transit time or time to diarrhea induced by castor oil. Consequent to the delay in gastric emptying, TMinh-23 administration significantly reduced the elevation in blood sugar in mice following an oral but not intraperitoneal glucose load. These results provide pharmacological evidence for involvement of TMEM16A in gastric emptying and suggest the utility of TMEM16A inhibition in disorders of accelerated gastric emptying, such as dumping syndrome, and potentially for improving glucose tolerance in diabetes mellitus/metabolic syndrome and enhancing satiety in obesity.-Cil, O., Anderson, M. O., Yen, R., Kelleher, B., Huynh, T. L., Seo, Y., Nilsen, S. P., Turner, J. R., Verkman, A. S. Slowed gastric emptying and improved oral glucose tolerance produced by a nanomolar-potency inhibitor of calcium-activated chloride channel TMEM16A.

Pairing MCL-1 inhibition with venetoclax improves therapeutic efficiency of BH3-mimetics in AML.

OBJECTIVES: Venetoclax combined with hypomethylating agents is a new therapeutic strategy frequently used for treating AML patients who are not eligible for conventional chemotherapy. However, high response rates are heterogeneous due to different mechanisms mediating resistance to venetoclax such as up-regulation of MCL-1 expression. We thus tested the anti-leukemic activity of S63845, a specific MCL-1 inhibitor. METHODS: Apoptosis induces by S63845 with or without venetoclax was evaluated in primary AML samples and in AML cell lines co-cultured or not with bone marrow (BM) mesenchymal stromal cells. Sensitivity of leukemic cells to S63845 was correlated to the expression level of BCL-2, MCL-1, and BCL-XL determined by Western Blot and mass spectrometry-based proteomics. RESULTS: We observed that even if MCL-1 expression is weak compared to BCL-2, S63845 induces apoptosis of AML cells and strongly synergizes with venetoclax. Furthermore, AML cells resistant to venetoclax are highly sensitive to S63845. Interestingly, the synergistic effect of S63845 toward venetoclax-mediated apoptosis of AML cells is still observed in a context of interaction with the BM microenvironment that intrinsically mediates resistance to BCL2 inhibition. CONCLUSION: These results are therefore of great relevance for clinicians as they provide the rational for combining BCL-2 and MCL-1 inhibition in AML.

Synthesis and Initial Biological Evaluation of Boron-Containing Prostate-Specific Membrane Antigen Ligands for Treatment of Prostate Cancer Using Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate-specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed a strong binding affinity to PSMA in a competition radioligand binding assay (IC50 from 555.7 to 20.3 nM). Three selected compounds 1a, 1d, and 1f were administered to mice, and their in vivo blocking of 68Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4-7 µg/g in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-l-phenylalanine (BPA). Taken together, these data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

CT can stratify patients as low risk for tibial neuropathy following a talus fracture.

OBJECTIVE: Determine the incidence of tibial neuropathy following talus fractures and CT's ability to stratify patients at risk for developing post-traumatic neuropathy. MATERIALS AND METHODS: In this IRB-approved retrospective analysis, 71 talus fractures and 8 contralateral control ankle CTs were reviewed by one observer blinded to clinical information. CT evidence suggestive of tibial neurovascular bundle injury included nerve displacement, perineural fat effacement/edema, and bone touching nerve. The association between these CT findings and clinically evident tibial neuropathy was analyzed. A semi-quantitative likelihood score was assigned based on the degree of the CT findings around the nerve. Interobserver agreement was calculated between 2 other readers. RESULTS: Twenty-five percent of patients in this cohort had clinical evidence of tibial neuropathy. There was a high specificity (0.87-0.93) and negative predictive value (0.83-0.87), a moderate accuracy (0.80-0.82), but a lower sensitivity (0.33-0.56) associated with the CT findings. Among the CT findings, nerve displacement (p < 0.0001) and bone touching nerve (p = 0.01) were associated with tibial neuropathy. A likelihood score of 2-5 was associated (p = 0.007-0.015) with tibial neuropathy. The presence of tibial neuropathy and nerve recovery were not associated with hospital length of stay, while CT findings were. There was substantial agreement between the three readers: likelihood scores 2+ (k = 0.78) and 3+ (k = 0.72). CONCLUSIONS: Tibial neuropathy occurs following talus fractures, and CT findings may help surgeons narrow down the number of patients requiring close neurological follow-up.

Vascular Cell Adhesion Molecule-Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques.

Atherosclerosis is a cardiovascular disease characterized by the formation of lipid-rich plaques within the walls of large arteries. Over time, a portion of these lesions can detach and lead to serious complications, such as strokes or heart attacks. Currently, there is no clinically effective way to detect the presence of atherosclerosis in patients until it has reached a relatively advanced stage. Furthermore, increasing evidence suggests that the pathobiological behavior of plaques is determined mainly by their composition, and not their size, which is the parameter usually monitored with current imaging techniques. In this work, we report protein-based agents that target the vascular cell adhesion molecule (VCAM1), a protein that plays a crucial role in atherosclerosis progression. In vivo experiments with murine atherosclerosis models indicated that the targeted protein nanoparticles were successful in detecting plaques of various sizes in the descending aorta and the aortic arch. This finding encourages the further development of these nanoscale agents for applications in the imaging, diagnosis, and treatment of cardiovascular diseases.

Therapeutic plasma exchange normalizes insulin-mediated response in a child with type 1 diabetes and insulin autoimmune syndrome.

BACKGROUND: Insulin autoimmune syndrome (IAS), characterized by glycemic dysregulation and life-threatening hypoglycemia, can occur in patients with type 1 diabetes (T1D). Diagnostic confirmation is complex but important in order to ensure timely initiation of definitive therapy. AIMS: We aimed to quantitate the degree of immunoglobulin-insulin complex (IIC) formation and its effects on glycemic control in a patient with T1D and IAS compared with T1D and non-T1D controls and before and after therapeutic plasma exchange (TPE). MATERIALS & METHODS: The prospective descriptive study was conducted between June 2015 and December 2015 in a quaternary children's hospital in Brisbane, Australia. Percent Free "Immunoreactive" Insulin (%FII) as assessed by polyethylene glycol precipitation studies and its relationship to plasma glucose and serum insulin concentration. RESULTS: Samples from the patient with T1D and IAS demonstrated lower mean %FII compared to T1D (23.8 ± 2.0 vs 52.0 ± 6.7; P < .0001) and non-T1D (23.8 ± 2.0 vs 102.9 ± 2.7; P < .0001) controls. This was associated with loss of glycemic predictability and frequent severe hypoglycemia. TPE increased %FII (23.8 ± 2.0 before TPE vs 83.6 ± 2.5 after TPE, P < .0001) and reestablished plasma glucose responsiveness to exogenous insulin. DISCUSSION: IAS should be considered in T1D patients with unexplained glycemic instability and hypoglycemia. The laboratory plays an integral diagnostic role. CONCLUSION: TPE is an effective method for removing IICs and normalizing insulin-mediated glucose responses.