α-Aryl substituted GdDOTA derivatives, the perfect contrast agents for MRI?

Enhancing the performance of Gd3+ chelates as relaxation agents for MRI has the potential to lower doses, improving safety and mitigating the environmental impact on our surface waters. More than three decades of research into manipulating the properties of Gd3+ have failed to develop a chelate that simultaneously optimizes all relevant parameters and affords maximal relaxivity. Introducing aryl substituents into the α-position of the pendant arms of a GdDOTA chelate affords chelates that, for the first time, simultaneously optimize all physico-chemical properties. Slowing tumbling by binding to human serum albumin affords a relaxivity of 110 ± 5 mM-1 s-1, close to the maximum possible. As discrete chelates, these α-aryl substituted GdDOTA chelates exhibit relaxivities that are 2-3 times higher than those of currently used agents, even at the higher fields (1.5 & 3.0 T) used in modern clinical MRI.

Diastereoselective Synthesis of α-Aryl-Substituted LnDOTA Chelates from Achiral Starting Materials by Deracemization Under Mild Conditions.

Substituted derivatives of the DOTA framework are of general interest to alter chelate properties and facilitate the conjugation of chelates to other molecular structures. However, the scope of substituents that can be introduced into the α-position has traditionally been limited by the availability of a suitable enantiopure starting materials to facilitate a stereoselective synthesis. Tetra-substituted DOTA derivatives with phenyl and benzoate substituents in the α-position have been prepared. Initial syntheses used enantiopure starting materials but did not afford enantiopure products. This indicates that the integrity of the stereocenters was not preserved during synthesis, despite the homo-chiral diastereoisomer being the major reaction product. The homochiral diastereoisomer could be produced as the major or sole reaction product when starting from racemic or even achiral materials. Deracemization was found to occur during chelation through the formation of an enolate stabilized by the aryl substituent. This general ability of aryl groups to enable deracemization greatly increases the range of substituents that can be introduced into DOTA-type ligands with diastereochemical selectivity.

A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.

Therapeutic neutralizing monoclonal antibody administration protects against lethal yellow fever virus infection.

Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.

Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity.

Hepatitis B virus has infected a third of the world's population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.

Analysis of the Relaxometric Properties of Extremely Rapidly Exchanging Gd3+ Chelates: Lessons from a Comparison of Four Isomeric Chelates.

Relaxometric analyses and in particular the use of fast-field cycling techniques have become routine in the study of paramagnetic metal complexes. The field dependence of the solvent proton relaxation properties (nuclear magnetic relaxation dispersion, NMRD) can provide unparalleled insights into the chemistry of these complexes. However, analyzing NMRD data is a multiparametric problem, and some sets of variables are mutually compensatory. Specifically, when fitting NMRD profiles, the metal-proton distance and the rotational correlation time constant have a push-pull relationship in which a change to one causes a predictable compensation in the other. A relaxometric analysis of four isomeric chelates highlights the pitfalls that await when fitting the NMRD profiles of chelates for which dissociative water exchange is extremely rapid. In the absence of independently verified values for one of these parameters, NMRD profiles can be fitted to multiple parameter sets. This means that NMRD fitting can inadvertently be used to buttress a preconceived notion of how the complex should behave when a different parameter set may more accurately describe the actual behavior. These findings explain why the effect of very rapid dissociative exchange on the hydration state of Gd3+ has remained obscured until only recently.

Differences in the Relaxometric Properties of Regioisomeric Benzyl-DOTA Bifunctional Chelators: Implications for Molecular Imaging.

The bifunctional chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane- N, N', N″, N‴-1,4,7,10-tetraacetate (IB-DOTA) is on paper the most attractive of the commercially available bifunctional chelators for magnetic resonance imaging (MRI) applications. The preserved DOTA scaffold is known to produce extremely kinetically and thermodynamically robust chelates with the Gd3+ ion. Also, ligation through four acetate pendant arms should ensure that the rapid water exchange kinetics so, crucial to the function of an MRI contrast agent are retained. However, upon ligation of the Gd3+ ion, IB-DOTA differentiates into two distinct isomers defined by the positions of the benzylic substituent (corner or side). A relaxometric analysis of these two isomers revealed marked differences in the property and behavior of the two chelates. Most notably the side isomer is found to be substantially more likely to aggregate in aqueous solution than its corner counterpart. This aggregation results in higher relaxivity for the side isomer versus the corner isomer, an observation that potentially obscures the impact of differences in water exchange kinetics between the two isomers. The side isomer is composed of a significant fraction of a twisted square antiprismatic coordination geometry that exchanges water more rapidly than optimal (τM = 7 ns) for maximizing relaxivity. The impact of this excessively fast exchange is not observed in the relaxivity of the side isomer only because in isolation this chelate tumbles much more slowly than the corner isomer. However, this situation is not expected to persist when the chelate is employed in a typical bioconjugate. These results imply that the corner isomer of IB-DOTA may represent a better choice of bifunctional chelator for bioconjugation applications in which a large macromolecule is to be tagged for MRI applications.

Accuracy and precision of age determination using growth layer groups for California sea lions (Zalophus californianus) with known ages.

Age determination from counts of growth layer groups (GLGs) in tooth dentine is a common method for aging marine mammals. Using known-aged animals, we validated this method for acid etched teeth of California sea lions (CSLs), Zalophus californianus. Between 1991 and 2013, the upper left canine (n = 33) was collected opportunistically during necropsy from animals tagged or branded as pups that later died. Overall, 55%-61% of age estimates by GLG counting were within 1 yr of the known-age in the sample of 1-30-yr-old CSLs. Accuracy of age estimates was found to be dependent on age of the CSLs, however. 71%-79% of age estimates were within 1 yr of the known-age in CSLs <10 yr old. These findings support the validity of counting GLGs to estimate age for CSLs <10 yr old to within 1 yr of accuracy.

HYPERMUCOVISCOUS KLEBSIELLA PNEUMONIAE ISOLATES FROM STRANDED AND WILD-CAUGHT MARINE MAMMALS OF THE US PACIFIC COAST: PREVALENCE, PHENOTYPE, AND GENOTYPE.

Emergent hypermucoviscous (HMV) strains of Klebsiella pneumoniae have been reported in multiple marine mammal species; however, there is limited information regarding the epidemiology and pathogenesis of this infection in these species. We determined the prevalence of HMV K. pneumoniae in wild-caught and stranded marine mammal populations on the US Pacific Coast. Samples were collected from 270 free-ranging California sea lions (CSLs; Zalophus californianus) captured at three discrete sampling sites and from 336 stranded marine mammals of various species. We recovered HMV K. pneumoniae only from CSLs, with a prevalence of 1.5% (4 of 275) in stranded animals, compared with 1.1% (3 of 270) in wild-caught animals. We assessed the phenotypic and genotypic variability of recovered HMV K. pneumoniae isolates recovered from CSLs ( n=11) and of archival HMV and non-HMV isolates from stranded marine mammals ( n=19). All but two HMV isolates were of the K2 serotype, whereas none of the non-HMV isolates belonged to this serotype. Of the HMV isolates, 96% (24 of 25) were PCR positive for the HMV-associated gene p- rmpA, whereas 92% (23 of 25) were PCR positive for p- rmpA2. Genetic fingerprinting by repetitive extragenic palindromic PCR showed four discrete clusters, demonstrating genotypic variability that loosely correlated with phenotype. Antimicrobial susceptibility testing revealed all isolates from stranded CSLs were susceptible to ceftiofur, indicating this antimicrobial agent is an appropriate choice for treatment of HMV K. pneumoniae infections in stranded CSLs. Our culture assay could reliably detect HMV K. pneumoniae from concentrations as low as 102 colony-forming units per milligram of feces. We identified the presence of HMV K. pneumoniae in both wild-caught and stranded CSLs from the US Pacific Coast and highlight the need for further studies to evaluate the potential impact of this pathogen on marine mammal health.

A 9-month-old boy with right-hand preference.

We present a case of a 9-month-old boy with a history of unequal hand movements, right-hand preference on physical examination, and cortical dysplasia on brain magnetic resonance imaging who was eventually diagnosed with cerebral palsy (CP). Although spasticity involving one or more limbs is the most common neurologic sign of CP, there can be a variety of other presenting symptoms including early hand preference, hypotonia, and oral motor dysfunction. In addition, traditional risk factors of premature birth and intrapartum complications are not always present. Given the wide clinical spectrum of CP, it is important for primary care providers to maintain a high index of suspicion to ensure appropriate diagnosis and early access to interventions.

Breakfast cereal sampling study for nutritional elements.

The National Institute of Standards and Technology (NIST) has established a Dietary Supplement Laboratory Quality Assurance Program (DSQAP) in collaboration with the National Institutes of Health Office of Dietary Supplements (NIH-ODS). The DSQAP invites laboratories twice annually to participate in interlaboratory studies where participants elect to measure concentrations of nutritional and/or toxic elements as well as active and/or marker compounds. One of these studies was designed to determine the effects of material granularity and sample processing techniques on measurement variability (precision) as well as to provide participating laboratories information on their performance relative to the NIST assigned values (bias) and to the other participants (concordance). Participants were asked to determine the mass fractions of Ca, Fe, and Zn, in mg/kg, in six breakfast cereal samples. Cereal samples consisted of three ground materials (homogenized wheat, wheat, and rice), two flake materials (wheat and rice) and a partially crushed material (a wheat/rice mixture). In general, approximately 25% of the laboratories processed and analyzed the suite of six cereal materials with adequate to exemplary measurement precision. Over half of the laboratories (60%) experienced measurement issues related to only a particular type of cereal matrix or for only a single element. A small number (15%) of laboratories experienced significant sample processing or measurement problems. Future studies planned by the DSQAP may be designed to use commercial products to aid laboratories with their sampling and analytical techniques.