Virilization of a 46,XX Fetus Following Aromatase Inhibitor Treatment of Breast Cancer.

Virilization of the 46,XX infant may be attributed to maternal or fetoplacental origin. Maternal sources may be endogenous, as with an androgen-producing tumor, or drug-related. Iatrogenic virilization by maternal drug exposure is rarely reported, with individual case reports and case series demonstrating the effects of progesterone and other medications affecting the pituitary-ovarian axis.1-3 The class of medications known as aromatase inhibitors are recognized as effective in treating hormone receptor-positive breast cancer by preventing the conversion of androgens into estrogens by aromatase. In fetal development, placental aromatase plays a critical role in preventing virilization of the XX fetus by maternal and fetal androgens during development. In the setting of placental aromatase deficiency, the XX fetus may be virilized. It is conceivable, therefore, that maternal exposure to aromatase inhibitors early in gestation may lead to in utero virilization, though there have been no known reports of this phenomenon to date. We present a case of virilization of a 46,XX infant attributed to pharmacologic aromatase inhibition. The infant's parents provided informed consent for the reporting of this case.

Risk of severe COVID-19 infection among adults with prior exposure to children.

Susceptibility and severity of COVID-19 infection vary widely. Prior exposure to endemic coronaviruses, common in young children, may protect against SARS-CoV-2. We evaluated risk of severe COVID-19 among adults with and without exposure to young children in a large, integrated healthcare system. Adults with children 0-5 years were matched 1:1 to adults with children 6-11 years, 12-18 years, and those without children based upon a COVID-19 propensity score and risk factors for severe COVID-19. COVID-19 infections, hospitalizations, and need for intensive care unit (ICU) were assessed in 3,126,427 adults, of whom 24% (N = 743,814) had children 18 years or younger, and 8.8% (N = 274,316) had a youngest child 0-5 years. After 1:1 matching, propensity for COVID-19 infection and risk factors for severe COVID-19 were well balanced between groups. Rates of COVID-19 infection were slightly higher for adults with exposure to older children (incident risk ratio, 1.09, 95% confidence interval, [1.05-1.12] and IRR 1.09 [1.05-1.13] for adults with children 6-11 and 12-18, respectively), compared to those with children 0-5 years, although no difference in rates of COVID-19 illness requiring hospitalization or ICU admission was observed. However, adults without exposure to children had lower rates of COVID-19 infection (IRR 0.85, [0.83-0.87]) but significantly higher rates of COVID-19 hospitalization (IRR 1.49, [1.29-1.73]) and hospitalization requiring ICU admission (IRR 1.76, [1.19-2.58]) compared to those with children aged 0-5. In a large, real-world population, exposure to young children was associated with less severe COVID-19 illness. Endemic coronavirus cross-immunity may play a role in protection against severe COVID-19.

Zonation in NASH - A key paradigm for understanding pathophysiology and clinical outcomes.

Non-alcoholic fatty liver disease (NAFLD) exists as a spectrum ranging from simple steatosis to histologically defined hepatocyte injury and inflammatory changes that define steatohepatitis (NASH), and increase risk for fibrosis. Although zonal differences in NASH have not been systematically studied, periportal involvement has been associated with worse metabolic outcomes and more hepatic fibrosis as compared to pericentral disease. These data suggest that hepatic zonation of disease may influence the diversity of clinical presentations. Similarly, several randomized clinical trials suggest a differential response based on zonation of disease, with preferential effects on periportal (cysteamine) or pericentral disease (obeticholic acid, pioglitazone). Intriguingly, morphogenic pathways known to affect zonal development and maintenance - WNT/ß-Catenin, Hedgehog, HIPPO/Yap/TAZ and Notch - have been implicated in NASH pathogenesis, and nuclear hormone receptors downstream of potential NASH therapeutics show zonal preferences. In this review, we summarize these data and propose that patient-specific activation of these pathways may explain the variability in clinical presentation, and the zone-specific response observed in clinical trials.

Targeting allostery in the Dynein motor domain with small molecule inhibitors.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

Analyzing Resistance to Design Selective Chemical Inhibitors for AAA Proteins.

Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them. Mutations that alter compound potency identify residues that make key interactions with the inhibitor and predict target-binding poses. Here, we apply this approach to analyze how diaminotriazole-based inhibitors bind spastin, a microtubule-severing AAA (ATPase associated with diverse cellular activities) protein. The distinct binding poses predicted for two similar inhibitors were confirmed by a series of X-ray structures. Importantly, our approach not only reveals how selective inhibition of the target can be achieved but also identifies resistance-conferring mutations at the early stages of the design process.

Using chemical inhibitors to probe AAA protein conformational dynamics and cellular functions.

The AAA proteins are a family of enzymes that play key roles in diverse dynamic cellular processes, ranging from proteostasis to directional intracellular transport. Dysregulation of AAA proteins has been linked to several diseases, including cancer, suggesting a possible therapeutic role for inhibitors of these enzymes. In the past decade, new chemical probes have been developed for AAA proteins including p97, dynein, midasin, and ClpC1. In this review, we discuss how these compounds have been used to study the cellular functions and conformational dynamics of AAA proteins. We discuss future directions for inhibitor development and early efforts to utilize AAA protein inhibitors in the clinical setting.

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity.

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

Incorporating the principles of the patient-centered medical home into a student-run free clinic.

As the health care delivery landscape changes, medical schools must develop creative strategies for preparing future physicians to provide quality care in this new environment. Despite the growing prominence of the patient-centered medical home (PCMH) as an effective model for health care delivery, few medical schools have integrated formal education on the PCMH into their curricula. Incorporating the PCMH model into medical school curricula is important to ensure that students have a comprehensive understanding of the different models of health care delivery and can operate effectively as physicians. The authors provide a detailed description of the process by which the Weill Cornell Community Clinic (WCCC), a student-run free clinic, has integrated PCMH principles into a service-learning initiative. The authors assessed patient demographics, diagnoses, and satisfaction along with student satisfaction. During the year after a PCMH model was adopted, 112 students and 19 licensed physicians volunteered their time. A review of the 174 patients seen from July 2011 to June 2012 found that the most common medical reasons for visits included management of hypertension, hyperlipidemia, diabetes, gastrointestinal conditions, arthritis, anxiety, and depression. During the year after the adoption of the PCMH model, 87% were very or extremely satisfied with their care, and 96% of the patients would recommend the WCCC to others. Students who participate in the WCCC gain hands-on experience in coordinating care, providing continuity of care, addressing issues of accessibility, and developing quality and safety metrics. The WCCC experience provides an integrative model that links service-learning with education on health care delivery in a primary care setting. The authors propose that adoption of this approach by other student-run clinics provides a substantial opportunity to improve medical education nationwide and better prepare future physicians to practice within this new model of health care delivery.

Direct kinetochore-spindle pole connections are not required for chromosome segregation.

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

A biomolecule-compatible visible-light-induced azide reduction from a DNA-encoded reaction-discovery system.

Using a system that accelerates the serendipitous discovery of new reactions by evaluating hundreds of DNA-encoded substrate combinations in a single experiment, we explored a broad range of reaction conditions for new bond-forming reactions. We discovered reactivity that led to a biomolecule-compatible, Ru(II)-catalysed azide-reduction reaction induced by visible light. In contrast to current azide-reduction methods, this reaction is highly chemoselective and is compatible with alcohols, phenols, acids, alkenes, alkynes, aldehydes, alkyl halides, alkyl mesylates and disulfides. The remarkable functional group compatibility and mild conditions of the reaction enabled the azide reduction of nucleic acid and oligosaccharide substrates, with no detectable occurrence of side reactions. The reaction was also performed in the presence of a protein enzyme without the loss of enzymatic activity, in contrast to two commonly used azide-reduction methods. The visible-light dependence of this reaction provides a means of photouncaging functional groups, such as amines and carboxylates, on biological macromolecules without using ultraviolet irradiation.

Free-solution oligonucleotide separation in nanoscale channels.

In this paper, we report an experimental study of electrokinetic transport and separation of double-stranded deoxyribonucleic acid (dsDNA) oligonucleotides in custom-fabricated fused-silica nanochannels filled with a gel-free sodium borate aqueous buffer. Mixtures of fluorescently labeled dsDNA molecules in the range of 10-100 base pair (bp), fluorescein, and fluorescein-12-UTP (UTP) were separated in less than 120 s in channels of depth ranging from 40 to 1560 nm. We varied the channel depth and background buffer concentration to achieve a 0.006-0.2 range of Debye length-to-channel-half-depth ratio (lambdaD/h), and a 0.004-1.7 range of the ratio of length of dsDNA molecule to channel half-depth (l/h). We find observed oligonucleotide migration times depend on both l/h and lambdaD/h. Electrophoretic mobility estimates agree well with published (micrometer-scale channel) values for background electrolyte (BGE) concentrations greater than approximately 10 mM. At BGE concentrations of 1 and 5 mM, mobility estimates in our nanochannels are higher than published values. Of the cases studied, the highest separation sensitivities were achieved in 100 nm channels with 1-10 mM ion density buffers. Potential applications of this technology include rapid small-scale sequencing and other fluorescence-based oligonucleotide separation and detection assays.

Granzyme B is dispensable for immunologic tolerance to self in a murine model of systemic lupus erythematosus.

OBJECTIVE: Proteolytic autoantigen cleavage by the serine protease granzyme B has been implicated in the development of systemic autoimmune disease; however, there has been no conclusive demonstration of a pathogenic role for granzyme B in autoimmunity. In this study, we evaluated the role of granzyme B in a murine model of autoimmunity. METHODS: To identify potential novel granzyme B substrates, complementary DNAs encoding nuclear factor 45 (NF45) and NF90 were used to generate (35)S-methionine-labeled proteins by coupled in vitro transcription/translation. Radiolabeled proteins were then incubated with purified recombinant granzyme B or caspases, and the cleavage products were analyzed by autoradiography. We also immunized granzyme B-deficient and granzyme B-intact mice with the mineral oil pristane. Production of autoantibodies directed against granzyme B substrates in response to pristane was evaluated by Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay. RESULTS: The double-stranded RNA-binding protein NF90 was identified as a novel substrate for caspases and granzyme B, both in vitro and in vivo. NF90 is uniquely cleaved by granzyme B in vitro; however, pristane immunization still induced anti-NF90 antibodies in granzyme B-deficient mice. Pristane-treated granzyme B-deficient mice also produced antibodies directed against the U1-70-kd antigen, a previously identified granzyme B substrate. Last, antibodies directed against U1-70 kd arose spontaneously in granzyme B-deficient mice. CONCLUSION: These results demonstrate that granzyme B is not required for the production of autoantibodies directed against antigens that are granzyme B substrates in vitro. The data also suggest a protective role for this proapoptotic protease in systemic autoimmunity.