Adaptive coding of stimuli is well documented in perception, where it supports efficient encoding over a broad range of possible percepts. Recently, a similar neural mechanism has been reported also in value-based decision, where it allows optimal encoding of vast ranges of values in PFC: neuronal response to value depends on the choice context (relative coding), rather than being invariant across contexts (absolute coding). Additionally, value learning is sensitive to the amount of feedback information: providing complete feedback (both obtained and forgone outcomes) instead of partial feedback (only obtained outcome) improves learning. However, it is unclear whether relative coding occurs in all PFC regions and how it is affected by feedback information. We systematically investigated univariate and multivariate feedback encoding in various mPFC regions and compared three modes of neural coding: absolute, partially-adaptive and fully-adaptive.

Twenty-eight human participants (both sexes) performed a learning task while undergoing fMRI scanning. On each trial, they chose between two symbols associated with a certain outcome. Then, the decision outcome was revealed. Notably, in one-half of the trials participants received partial feedback, whereas in the other half they got complete feedback. We used univariate and multivariate analysis to explore value encoding in different feedback conditions.

We found that both obtained and forgone outcomes were encoded in mPFC, but with opposite sign in its ventral and dorsal subdivisions. Moreover, we showed that increasing feedback information induced a switch from absolute to relative coding. Our results suggest that complete feedback information enhances context-dependent outcome encoding.

SIGNIFICANCE STATEMENT This study offers a systematic investigation of the effect of the amount of feedback information (partial vs complete) on univariate and multivariate outcome value encoding, within multiple regions in mPFC and cingulate cortex that are critical for value-based decisions and behavioral adaptation. Moreover, we provide the first comparison of three possible models of neural coding (i.e., absolute, partially-adaptive, and fully-adaptive coding) of value signal in these regions, by using commensurable measures of prediction accuracy. Taken together, our results help build a more comprehensive picture of how the human brain encodes and processes outcome value. In particular, our results suggest that simultaneous presentation of obtained and foregone outcomes promotes relative value representation.

Giving birth triggers a wide repertoire of physiological and behavioral changes in the mother to enable her to feed and care for her offspring. These changes require coordination and are often orchestrated from the CNS, through as of yet poorly understood mechanisms. A neuronal population with a central role in puerperal changes is the tuberoinfundibular dopamine (TIDA) neurons that control release of the pituitary hormone, prolactin, which triggers key maternal adaptations, including lactation and maternal care. Here, we used Ca²⁺ imaging on mice from both sexes and whole-cell recordings on female mouse TIDA neurons in vitro to examine whether they adapt their cellular and network activity according to reproductive state. In the high-prolactin state of lactation, TIDA neurons shift to faster membrane potential oscillations, a reconfiguration that reverses upon weaning. During the estrous cycle, however, which includes a brief, but pronounced, prolactin peak, oscillation frequency remains stable. An increase in the hyperpolarization-activated mixed cation current, I_h, possibly through unmasking as dopamine release drops during nursing, may partially explain the reconfiguration of TIDA rhythms. These findings identify a reversible plasticity in hypothalamic network activity that can serve to adapt the dam for motherhood.

SIGNIFICANCE STATEMENT Motherhood requires profound behavioral and physiological adaptations to enable caring for offspring, but the underlying CNS changes are poorly understood. Here, we show that, during lactation, neuroendocrine dopamine neurons, the "TIDA" cells that control prolactin secretion, reorganize their trademark oscillations to discharge in faster frequencies. Unlike previous studies, which typically have focused on structural and transcriptional changes during pregnancy and lactation, we demonstrate a functional switch in activity and one that, distinct from previously described puerperal modifications, reverses fully on weaning. We further provide evidence that a specific conductance (I_h) contributes to the altered network rhythm. These findings identify a new facet of maternal brain plasticity at the level of membrane properties and consequent ensemble activity.

Biases in sensory perception can arise from both experimental manipulations and personal trait-like features. These idiosyncratic biases and their neural underpinnings are often overlooked in studies on the physiology underlying perception. A potential candidate mechanism reflecting such idiosyncratic biases could be spontaneous alpha band activity, a prominent brain rhythm known to influence perceptual reports in general. Using a temporal order judgment task, we here tested the hypothesis that alpha power reflects the overcoming of an idiosyncratic bias. Importantly, to understand the interplay between idiosyncratic biases and contextual (temporary) biases induced by experimental manipulations, we quantified this relation before and after temporal recalibration. Using EEG recordings in human participants (male and female), we find that prestimulus frontal alpha power correlates with the tendency to respond relative to an own idiosyncratic bias, with stronger α leading to responses matching the bias. In contrast, alpha power does not predict response correctness. These results also hold after temporal recalibration and are specific to the alpha band, suggesting that alpha band activity reflects, directly or indirectly, processes that help to overcome an individual's momentary bias in perception. We propose that combined with established roles of parietal α in the encoding of sensory information frontal α reflects complementary mechanisms influencing perceptual decisions.

SIGNIFICANCE STATEMENT The brain is a biased organ, frequently generating systematically distorted percepts of the world, leading each of us to evolve in our own subjective reality. However, such biases are often overlooked or considered noise when studying the neural mechanisms underlying perception. We show that spontaneous alpha band activity predicts the degree of biasedness of human choices in a time perception task, suggesting that alpha activity indexes processes needed to overcome an individual's idiosyncratic bias. This result provides a window onto the neural underpinnings of subjective perception, and offers the possibility to quantify or manipulate such priors in future studies.

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU⁺ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.

SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1. In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1. These findings are the first to reveal a role for Neurog2 in hypothalamic development.

One of the first signs of viral infection is body-wide aches and pain. Although this type of pain usually subsides, at the extreme, viral infections can induce painful neuropathies that can last for decades. Neither of these types of pain sensitization is well understood. A key part of the response to viral infection is production of interferons (IFNs), which then activate their specific receptors (IFNRs) resulting in downstream activation of cellular signaling and a variety of physiological responses. We sought to understand how type I IFNs (IFN-α and IFN-β) might act directly on nociceptors in the dorsal root ganglion (DRG) to cause pain sensitization. We demonstrate that type I IFNRs are expressed in small/medium DRG neurons and that their activation produces neuronal hyper-excitability and mechanical pain in mice. Type I IFNs stimulate JAK/STAT signaling in DRG neurons but this does not apparently result in PKR-eIF2α activation that normally induces an anti-viral response by limiting mRNA translation. Rather, type I IFNs stimulate MNK-mediated eIF4E phosphorylation in DRG neurons to promote pain hypersensitivity. Endogenous release of type I IFNs with the double-stranded RNA mimetic poly(I:C) likewise produces pain hypersensitivity that is blunted in mice lacking MNK-eIF4E signaling. Our findings reveal mechanisms through which type I IFNs cause nociceptor sensitization with implications for understanding how viral infections promote pain and can lead to neuropathies.

SIGNIFICANCE STATEMENT It is increasingly understood that pathogens interact with nociceptors to alert organisms to infection as well as to mount early host defenses. Although specific mechanisms have been discovered for diverse bacterial and fungal pathogens, mechanisms engaged by viruses have remained elusive. Here we show that type I interferons, one of the first mediators produced by viral infection, act directly on nociceptors to produce pain sensitization. Type I interferons act via a specific signaling pathway (MNK-eIF4E signaling), which is known to produce nociceptor sensitization in inflammatory and neuropathic pain conditions. Our work reveals a mechanism through which viral infections cause heightened pain sensitivity

The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.

SIGNIFICANCE STATEMENT The AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.

Hydrocephalus is a pathologic condition associated with various brain diseases, including Alzheimer's disease (AD). Dysfunctional ependymal cells (EpCs) are believed to contribute to the development of hydrocephalus. It is thus of interest to investigate EpCs' development and function. Here, we report that vacuolar protein sorting-associated protein 35 (VPS35) is critical for EpC differentiation, ciliogenesis, and survival, and thus preventing neonatal hydrocephalus. VPS35 is abundantly expressed in EpCs. Mice with conditional knock-out (cKO) of Vps35 in embryonic (Vps35^GFAP-Cre and Vps35^Emx1-Cre) or postnatal (Vps35^Foxj1-CreER) EpC progenitors exhibit enlarged lateral ventricles (LVs) and hydrocephalus-like pathology. Further studies reveal marked reductions in EpCs and their cilia in both Vps35^GFAP-Cre and Vps35^Foxj1-CreER mutant mice. The reduced EpCs appear to be due to impairments in EpC differentiation and survival. Additionally, both Vps35^GFAP-Cre and Vps35^Foxj1-CreER neonatal pups exhibit increased cell proliferation and death largely in a region close to LV-EpCs. Many microglia close to the mutant LV-EpC region become activated. Depletion of the microglia by PLX3397, an antagonist of colony-stimulating factor 1 receptor (CSF1R), restores LV-EpCs and diminishes the pathology of neonatal hydrocephalus in Vps35^Foxj1-CreER mice. Taken together, these observations suggest unrecognized functions of Vps35 in EpC differentiation, ciliogenesis, and survival in neonatal LV, and reveal pathologic roles of locally activated microglia in EpC homeostasis and hydrocephalus development.

SIGNIFICANCE STATEMENT This study reports critical functions of vacuolar protein sorting-associated protein 35 (VPS35) not only in promoting ependymal cell (EpC) differentiation, ciliogenesis, and survival, but also in preventing local microglial activation. The dysfunctional EpCs and activated microglia are likely to induce hydrocephalus.

Awareness for surprising sensory events is shaped by prior belief inferred from past experience. Here, we combined hierarchical Bayesian modeling with fMRI on an associative learning task in 28 male human participants to characterize the effect of the prior belief of tactile events on connections mediating the outcome of perceptual decisions. Activity in anterior insular cortex (AIC), premotor cortex (PMd), and inferior parietal lobule (IPL) were modulated by prior belief on unexpected targets compared with expected targets. On expected targets, prior belief decreased the connection strength from AIC to IPL, whereas it increased the connection strength from AIC to PMd when targets were unexpected. Individual differences in the modulatory strength of prior belief on insular projections correlated with the precision that increases the influence of prediction errors on belief updating. These results suggest complementary effects of prior belief on insular-frontoparietal projections mediating the precision of prediction during probabilistic tactile learning.

SIGNIFICANCE STATEMENT In a probabilistic environment, the prior belief of sensory events can be inferred from past experiences. How this prior belief modulates effective brain connectivity for updating expectations for future decision-making remains unexplored. Combining hierarchical Bayesian modeling with fMRI, we show that during tactile associative learning, prior expectations modulate connections originating in the anterior insula cortex and targeting salience-related and attention-related frontoparietal areas (i.e., parietal and premotor cortex). These connections seem to be involved in updating evidence based on the precision of ascending inputs to guide future decision-making.

Autism spectrum disorder (ASD) is characterized partly by atypical attentional engagement, reflected in exaggerated and variable responses to sensory stimuli. Attentional engagement is known to be regulated by the locus ceruleus (LC). Moderate baseline LC activity globally dampens neural responsivity and is associated with adaptive deployment and narrowing of attention to task-relevant stimuli. In contrast, increased baseline LC activity enhances neural responsivity across cortex and widening of attention to environmental stimuli regardless of their task relevance. Given attentional atypicalities in ASD, this study is the first to evaluate whether, under different attentional task demands, individuals with ASD exhibit a different profile of LC activity compared with typically developing controls. Males and females with ASD and age- and gender-matched controls participated in a one-back letter detection test while task-evoked pupillary responses, an established correlate for LC activity, were recorded. Participants completed this task in two conditions, either in the absence or presence of distractor auditory tones. Compared with controls, individuals with ASD evinced atypical pupillary responses in the presence versus absence of distractors. Notably, this atypical pupillary profile was evident despite the fact that both groups exhibited equivalent task performance. Moreover, between-group differences in pupillary responses were observed specifically in response to task-relevant events, providing confirmation that the group differences most likely were specifically associated with distinctions in LC activity. These findings suggest that individuals with ASD show atypical modulation of LC activity with changes in attentional demands, offering a possible mechanistic and neurobiological account for attentional atypicalities in ASD.

SIGNIFICANCE STATEMENT Individuals with autism spectrum disorder (ASD) exhibit atypical attentional behaviors, including altered sensory responses and atypical fixedness, but the neural mechanism underlying these behaviors remains elusive. One candidate mechanism is atypical locus ceruleus (LC) activity, as the LC plays a critical role in attentional modulation. Specifically, LC activity is involved in regulating the trade-off between environmental exploration and focused attention. This study shows that, under tightly controlled conditions, task-evoked pupil responses, an LC activity proxy, are lower in individuals with ASD than in controls, but only in the presence of task-irrelevant stimuli. This suggests that individuals with ASD evince atypical modulation of LC activity in accordance with changes in attentional demands, offering a mechanistic account for attentional atypicalities in ASD.

Calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, are widely used as standard immunosuppressants in organ transplantation recipients. However, these drugs can cause severe pain in patients, commonly referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin inhibition increases NMDAR activity in the spinal cord, the underlying mechanism remains enigmatic. Using an animal model of CIPS, we found that systemic administration of FK506 in male and female mice significantly increased the amount of α2-1–GluN1 complexes in the spinal cord and the level of α2-1–bound GluN1 proteins in spinal synaptosomes. Treatment with FK506 significantly increased the frequency of mEPSCs and the amplitudes of monosynaptic EPSCs evoked from the dorsal root and puff NMDAR currents in spinal dorsal horn neurons. Inhibiting α2-1 with gabapentin or disrupting the α2-1–NMDAR interaction with α2-1Tat peptide completely reversed the effects of FK506. In α2-1 gene KO mice, treatment with FK506 failed to increase the frequency of NMDAR-mediated mEPSCs and the amplitudes of evoked EPSCs and puff NMDAR currents in spinal dorsal horn neurons. Furthermore, systemic administration of gabapentin or intrathecal injection of α2-1Tat peptide reversed thermal and mechanical hypersensitivity in FK506-treated mice. In addition, genetically deleting GluN1 in dorsal root ganglion neurons or α2-1 genetic KO similarly attenuated FK506-induced thermal and mechanical hypersensitivity. Together, our findings indicate that α2-1–bound NMDARs mediate calcineurin inhibitor-induced tonic activation of presynaptic and postsynaptic NMDARs at the spinal cord level and that presynaptic NMDARs play a prominent role in the development of CIPS.

SIGNIFICANCE STATEMENT Calcineurin inhibitors are immunosuppressants used to prevent rejection of transplanted organs and tissues. However, these drugs can cause severe, unexplained pain. We showed that calcineurin inhibition enhances physical interaction between α2-1 and NMDARs and their synaptic trafficking in the spinal cord. α2-1 is essential for calcineurin inhibitor-induced aberrant activation of presynaptic and postsynaptic NMDARs in the spinal cord. Furthermore, inhibiting α2-1 or disrupting α2-1–NMDAR interaction reduces calcineurin inhibitor-induced pain hypersensitivity. Eliminating NMDARs in primary sensory neurons or α2-1 KO also attenuates calcineurin inhibitor-induced pain hypersensitivity. This new information extends our mechanistic understanding of the role of endogenous calcineurin in regulating synaptic plasticity and nociceptive transmission and suggests new strategies for treating this painful condition.

As the clock ticks on the Millennium Development Goals (MDGs), the world community is deep in discussion over the successor global framework. Many current practices are damaging the planet’s ecosystems and the biodiversity essential for healthy food production. By 2050 an estimated additional 2 billion people will be living on Earth.  This means food production must rise by 60%. From 8 MDGs [...]

Fish plays an important role in fighting hunger and malnutrition and is the main source of animal protein in many developing countries. Seafood is not only a source of proteins and healthy long-chain omega-3 fats, but also an essential source of other nutrients like iodine, vitamin D and calcium, which are crucial to living a healthy life. Here are 10 interesting [...]

Ecosystem services make human life possible by, for example, providing nutritious food and clean water, regulating disease and climate, supporting the pollination of crops and soil formation, and providing recreational, cultural and spiritual benefits. In 2014, the value of ecosystem services was estimated at a staggering US$ 125 trillion.  Ecosystem services, provided by biodiversity, are fundamental to food production and [...]

Climate change is what most of us perceive as the top global threat, and the dangers it poses affect present and future generations alike.  How global warming is threatening the planet has been a theme in children’s books for all ages for some time.   How everyone, especially today’s youth, can make a difference to the future of the world [...]

The oceans have it all: from microscopic life to the largest animal that has ever lived on Earth, from the colourless to the shimmering, from the frozen to the boiling and from the sunlit to the mysterious dark of the deepest parts of the planet. Oceans are an essential component of the Earth's ecosystem -- a source of biodiversity, food, and [...]

Perhaps from outer space, it is easy to get the perspective that we only have one earth and that it is succumbing to climate change. Seeing the earth from space though is a feat that, unfortunately, most of us will never accomplish. We have to rely on Astronaut Thomas Pesquet and the other brave women and men astronauts to provide [...]

The digital revolution has changed the way we work, access information and connect with each other. It offers opportunities to those who can use the new technologies, but also presents new challenges for those who are left behind. Often referred to collectively as Information and Communications Technologies or ICTs, these technologies are any method of electronically sharing or storing data: telephones, [...]

The National Cherry Blossom Festival has moved online due to the novel coronavirus pandemic

‘What on earth were they doing there?’ one researcher asks

Mary Pearson, who was briefly engaged to the writer's brother, may be the real-life counterpart of Lydia Bennet from "Pride and Prejudice"

The prickly perch is an exciting sign of success for the birds, which came off the endangered species list in 2007

Scientists discover neurotoxin-producing bacteria living on the skin of rough-skinned newts

The 18th-century Edinburgh factory once produced a million bottles a week

The simultaneous mating calls of the male pug-nosed tree frog confuses bats but not female frogs

Items recovered include fossils, paintings, ancient coins, ceramics and jewelry

Graffiti artists and muralists are sending messages of hope and despair with coronavirus public art

An abstract aerial view of a sand dune at sunset at Imperial Sand Dunes, Glamis, California.

While COVID-19 has us homebound, it’s a good time to reflect on the peculiar histories of housewares we take for granted

A filmed documentary on the University of Michigan 1970 environmental teach-in, asks the questions: Do teach-ins work? (The Bentley Historical Library at the University of Michigan)

In a written decision released Thursday, a panel of three appeal court judges said the judge who granted the award to McCaw North Drilling and Blasting Ltd. misinterpreted a clause in the contract for the cleanup.

The N.W.T. and Nunavut Workers’ Safety and Compensation Commission says it filed multiple charges in the Nunavut Court of Justice under the Nunavut Safety Act on May 1.

The government of the Northwest Territories has made an exemption on border restrictions for a veterinarian from Alberta to practice in the territory, after he received public and political support.

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The head of a Sudbury-based book publishing company says she’s shifting launches online due to COVID-19.

The following is a guest post by Martha H. Kennedy, Curator of Popular & Applied Graphic Art, Prints & Photographs Division. During Will Eisner Week, March 1-7, 2020, the Library of Congress joins art schools, libraries, universities, and museums in a global celebration of this legendary creator’s contributions to the world of comic art and […]

Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method can be applied for use of adherent or suspension cells and can significantly reduce nonspecific background in an immunoprecipitation by separation of cellular compartments into individual fractions. The lysis of cells by differential detergents permits the rapid extraction of proteins from the cytoplasm (digitonin), the cytoplasmic membranes, and organelles (Triton X-100), and nucleoplasm (Tween/DOC), facilitated through the use of distinct extraction buffers. Cytoplasmic and nuclear matrix proteins as well as DNA are left behind during the detergent-based extraction.

In this protocol, DNA fragments are separated according to size by electrophoresis through low-melting-temperature agarose, and then recovered by melting the agarose and extracting with phenol:chloroform. The protocol works best for DNA fragments ranging in size from 0.5 to 5.0 kb. Yields of DNA fragments outside this range are usually lower, but often are sufficient for many purposes.

Successful modification of the bacterial artificial chromosome (BAC) after two-step BAC engineering is confirmed in two separate polymerase chain reactions (PCRs). The first reaction (5' co-integrate PCR) uses a forward 5' co-integrate primer (a sequence located upstream of the 5' end of the A-box) and a reverse 3' primer on the vector (175PA+50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3' co-integrate PCR) uses a forward 5' primer on the recA gene (RecA1300S) and a reverse 3' co-integrate primer (a sequence located downstream from the 3' end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including sacB and recA genes become UV light sensitive.

Bacterial artificial chromosome (BAC) clones are rendered electrocompetent and transformed with the recombinant shuttle vector, pLD53SCAB/AB-box. Cointegrates are selected by growth on chloramphenicol and ampicillin to ensure recombination of the shuttle vector into the BAC.

Plasmid DNA is prepared from the recombinant shuttle vector pLD53.SCAB/A-B created by cloning of the A and B homology arms for two-step bacterial artificial chromosome (BAC) engineering. To confirm that the A-box and B-box arms have been successfully incorporated into pLD53.SCAB, the pattern of enzyme digestion of the modified plasmid is compared with that of the unmodified pLD53.SCAB. Once the shuttle vector is shown to carry the proper sequences, it is ready for transfer into the BAC host.

This protocol describes the preparation of the shuttle vector before its introduction into bacterial artificial chromosome (BAC) host cells for BAC two-step engineering. The homology arm sequences, prepared previously, are introduced by ligation into the digested shuttle vector DNA to provide sites for recombination within the BAC clone. Crude lysates of individual bacterial transformants serve as templates in polymerase chain reaction (PCR) analysis to confirm the presence of the homology arms in the recombinant shuttle vector.

The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector.

In two-step bacterial artificial chromosome (BAC) engineering, a single plasmid is introduced into the BAC-carrying cell lines. The shuttle vector pLD53.SCAB (or pLD53.SCAEB) carries the recA gene and the R6K origin, which requires the protein to replicate. PIR2 cells, expressing , are typically used for the amplification of the vector and maintain about 15 copies/cell of the donor vector, which is relatively stable in this host.

BIS Bulletin No 8, April 2020. Information on local labour markets and Google searches can be used to construct a measure of the vulnerability of employment in different regions of the United States to the Covid-19 shock. Regional exposure to Covid-19 varies significantly, ranging from a low of 2% to a high of 98% of total local employment. We test for the usefulness of the Covid-19 exposure measure by showing that areas with higher exposure report more Google search queries related to the pandemic and unemployment benefits.

BCBS Press release "Basel Committee sets out additional measures to alleviate the impact of Covid-19", 3 April 2020

It works like a very fine "molecular knob" able to modulate the electrical activity of the neurons of our cerebral cortex, crucial to the functioning of our brain.

Visit our four downtown locations where you can taste euphoria in our creative and delicious all natural ice-cream, yogurts and sorbets!

Bike the Sites of the Nation's Capital! It's the fun and easy way to get to get up close and personal with Washington's landmarks