Mapping uterine calcium dynamics during the ovulatory cycle in live mice.

Uterine contraction patterns vary during the ovulatory cycle and throughout pregnancy but prior measurements have produced limited and conflicting information on these patterns. We combined a virally delivered genetically encoded calcium reporter (GCaMP8m) and ultra-widefield imaging in live nonpregnant mice to characterize uterine calcium dynamics at organ scale throughout the estrous cycle. Prior to ovulation (proestrus and estrus) uterine excitations primarily initiated in a region near the oviduct, but after ovulation (metestrus and diestrus), excitations initiated at loci homogeneously distributed throughout the organ. The frequency of excitation events was lowest in proestrus and estrus, higher in metestrus and highest in diestrus. These results establish a platform for mapping uterine activity, and show that the question of whether there is an anatomically localized trigger for uterine excitations depends on the estrous cycle phase.

Validation of algorithms to identify adverse perinatal outcomes in the Medicaid Analytic Extract database.

BACKGROUND: The Medicaid Analytic eXtract (MAX) is a health care utilization database from publicly insured individuals that has been used for studies of drug safety in pregnancy. Claims-based algorithms for defining many important maternal and neonatal outcomes have not been validated. OBJECTIVE: To validate claims-based algorithms for identifying selected pregnancy outcomes in MAX using hospital medical records. METHODS: The medical records of mothers who delivered between 2000 and 2010 within a single large healthcare system were linked to their claims in MAX. Claims-based algorithms for placental abruption, preeclampsia, postpartum hemorrhage, small for gestational age, and noncardiac congenital malformation were defined. Fifty randomly sampled cases for each outcome identified using these algorithms were selected, and their medical records were independently reviewed by two physicians to confirm the presence of the diagnosis of interest; disagreements were resolved by a third physician reviewer. Positive predictive values (PPVs) and 95% confidence intervals (CIs) of the claims-based algorithms were calculated using medical records as the gold standard. RESULTS: The linked cohort included 10,899 live-birth pregnancies. The PPV was 92% (95% CI, 82%-97%) for placental abruption, 82% (95% CI, 70%-91%) for preeclampsia, 74% (95% CI, 61%-85%) for postpartum hemorrhage, 92% (95% CI, 82%-97%) for small for gestational age, and 86% (95% CI, 74%-94%) for noncardiac congenital malformation. CONCLUSIONS: Across the perinatal outcomes considered, PPVs ranged between 74% and 92%. These PPVs can inform bias analyses that correct for outcome misclassification.

Sphingomyelinase D inhibits store-operated Ca2+ entry in T lymphocytes by suppressing ORAI current.

Infections caused by certain bacteria including Mycobacterium tuberculosis and Corynebacterium pseudotuberculosis provoke inflammatory responses characterized by the formation of granulomas with necrotic foci-so-called caseous necrosis. The granulomas of infected animals show prominent infiltration by T lymphocytes, and T cell depletion increases host mortality. Notorious zoonotic C. pseudotuberculosis secretes sphingomyelinase (SMase) D, a phospholipase that cleaves off the choline moiety of sphingomyelin, a phospholipid found primarily in the outer leaflet of host cell plasma membranes. Experimental C. pseudotuberculosis strains that lack SMase D are markedly less infectious and unable to spread in hosts, indicating that this enzyme is a crucial virulence factor for sustaining the caseous lymphadenitis infections caused by this microbe. However, the molecular mechanism by which SMase D helps bacteria evade the host's immune response remains unknown. Here, we find that SMase D inhibits store-operated Ca(2+) entry (SOCE) in human T cells and lowers the production of the SOCE-dependent cytokines interleukin-2, which is critical for T cell growth, proliferation, and differentiation, and tumor necrosis factor α, which is crucial for the formation and maintenance of granulomas in microbial infections. SMase D inhibits SOCE through a previously unknown mechanism, namely, suppression of Orai1 current, rather than through altering gating of voltage-gated K(+) channels. This finding suggests that, whereas certain genetic mutations abolish Orai1 activity causing severe combined immunodeficiency (SCID), bacteria have the ability to suppress Orai1 activity with SMase D to create an acquired, chronic SCID-like condition that allows persistent infection. Thus, in an example of how virulence factors can disrupt key membrane protein function by targeting phospholipids in host cell membranes, our study has uncovered a novel molecular mechanism that bacteria can use to thwart host immunity.

Justice for the average Joe: the role of envy and the mentalizing network in the deservingness of others' misfortunes.

The misfortunes of enviable individuals are met by observers with pleasure whereas those of "average", non-enviable individuals elicit pain. These responses are mirrored in deservingness judgments, as enviable individuals' misfortunes are perceived as deserved and those of non-enviable individuals perceived as undeserved. However, the neural underpinnings of these deservingness disparities remain unknown. To explore this phenomenon, we utilized fMRI to test the hypotheses that (A) non-enviable targets' misfortunes would be associated with activation of brain regions that mediate empathic responding (pain matrix, mentalizing network) and not for enviable targets and (B) that activation of those regions would predict decreases in deservingness judgments. Supporting our first hypothesis, the misfortunes of non-enviable targets (as opposed to good fortunes) were associated with activation of the mentalizing network: medial prefrontal cortex, posterior cingulate cortex, temporal-parietal junction, and anterior temporal lobes. Supporting our second hypothesis, dorsomedial prefrontal cortex activation from this contrast was negatively correlated with subsequent reports of how much the non-enviable target deserved his/her misfortune. These findings suggest that non-enviable individuals' misfortunes are perceived as unjust due, in part, to the recruitment of the mentalizing network.

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase.

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K(+) (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids' interactions with voltage sensors. Then, using SMase D to probe lipid-channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na(+) (Na(V)) and Ca(2+) channels but also markedly slows Na(V) channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native K(V)1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native Na(V) channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

Post-translational regulation and nuclear entry of TIMELESS and PERIOD are affected in new timeless mutant.

The molecular circadian clock consists of a feedback loop in which canonical clock proteins negatively regulate transcription of their own genes. Timed nuclear entry of these proteins is critical, but regulation of this event is poorly understood. In Drosophila melanogaster, the idea that nuclear entry of PERIOD (PER) is controlled by its partner protein TIMELESS (TIM) has been challenged by several studies. We identify here a novel mutation in the tim gene that eliminates behavioral rhythms while allowing robust expression of TIM and PER. Mutant TIM can bind to and stabilize PER. However, neither protein is expressed cyclically, and phosphorylation of both is reduced. In addition, TIM and PER are localized in the cytoplasm at all times of day, and mutant TIM attenuates transcriptional feedback by PER in cultured cells, suggesting that it holds PER in the cytoplasm. In fact, much of the reduced phosphorylation of PER in the new tim mutant appears to result from the cytoplasmic localization of PER. Interestingly, mutating a threonine near the original mutation produces similar phenotypes, raising the possibility that defective phosphorylation is the basis of TIM dysfunction in the novel tim mutant. We also show that a stable form of PER is cytoplasmic in tim-null flies. These studies establish an essential role of TIM in the timed nuclear entry of PER.

Uncovering genetic relationships using small molecules that selectively target yeast cell cycle mutants.

Genetic analysis in budding yeast has shown that multiple G1 cyclins and cyclin-dependent kinases control cell cycle entry, polarized growth, and spindle pole duplication. The G1 cyclins Cln1 and Cln2 associate with the cyclin-dependent kinase Cdc28 to facilitate cell cycle progression and development of the cleavage apparatus. We have developed a chemical genetic approach toward the discovery of compounds that target G1 control pathways by screening for compounds that selectively kill a yeast strain lacking the G1 cyclins Cln1 and Cln2. A class of small molecules was identified that is highly toxic toward the cln1 Delta cln2 Delta double mutant and has relatively little effect on wild-type yeast. We call these compounds 'clinostatins' for their selectivity toward the cln1/2 deletion strain. Clinostatins were used in a genome-wide chemical synthetic lethality screen to identify other genes required for growth in the presence of the drug. Other deletions that were sensitive to the drug include members of the protein kinase C(PKC)-dependent MAP kinase pathway. These results suggest an approach for combining chemical synthetic lethality and chemical genomic screens to uncover novel genetic interactions that can be applied to other eukaryotic pathways of interest.

Extended peptoids: a new class of oligomers based on aromatic building blocks.

Peptoids (N-substituted polyglycines) represent a class of bioinspired oligomers that have unique physical and structural properties. Here we report the construction of "extended peptoids" based on aromatic building blocks, in which the N-alkylaminoacetyl group of the peptoid backbone has been replaced by an N-alkylaminomethylbenzoyl spacer. Both meta- and para-bromomethylbenzoic acids were synthesized, providing access to a new class of peptoids. Further, inclusion of hydrophilic side chains confers water solubility to these compounds, showing that, like simple peptoids, extended peptoids add an extra dimension to synthetic polyamide oligomers with potential application in a variety of biological contexts.