Anatomical restructuring of a lateralized neural circuit during associative learning by asymmetric insulin signaling.

Studies of neuronal connectivity in model organisms, i.e., of their connectomes, have been instrumental in dissecting the structure-function relationship of nervous systems. However, the limited sample size of these studies has impeded analyses into how variation of connectivity across populations may influence circuit architecture and behavior. Moreover, little is known about how experiences induce changes in circuit architecture. Here, we show that an asymmetric salt-sensing circuit in the nematode Caenorhabditis elegans exhibits variation that predicts the animals' salt preferences and undergoes restructuring during salt associative learning. Naive worms memorize and prefer the salt concentration they experience in the presence of food through a left-biased neural network architecture. However, animals conditioned at elevated salt concentrations change this left-biased network to a right-biased network. This change in circuit architecture occurs through the addition of new synapses in response to asymmetric, paracrine insulin signaling. Therefore, experience-dependent changes in an animal's neural connectome are induced by insulin signaling and are fundamental to learning and behavior.

Restructuring of an asymmetric neural circuit during associative learning.

Asymmetric brain function is common across the animal kingdom and involved in language processing, and likely in learning and memory. What regulates asymmetric brain function remains elusive. Here, we show that the nematode Caenorhabditis elegans restructures an asymmetric salt sensing neural circuit during associative learning. Worms memorize and prefer the salt concentration at which they were raised in the presence of food through a left-biased network architecture. When conditioned at elevated salt concentrations, animals change the left-biased to a right-biased network, which explains the changed salt-seeking behavior. The changes in circuit architecture require new synapse formation induced through asymmetric, paracrine insulin-signaling. Therefore, experience-dependent changes in asymmetric network architecture rely on paracrine insulin signaling and are fundamental to learning and behavior.

Interleaved NQR detection using atomic magnetometers.

Interleaved Nuclear Quadrupole Resonance (NQR) detection was conducted on ammonium nitrate and potassium chlorate using two 87Rb magnetometers, where potassium chlorate is measured during the T1 limited recovery time of ammonium nitrate. The multi-pass magnetometers are rapidly matched to the NQR frequencies, 531 kHz and 423 kHz, with the use of a single tuning field. For ease of implementation, a double resonant tank circuit was used for excitation, but could be replaced by a broad-band transmitter. All work was done in an unshielded environment and compared to conventional coil detection. The two magnetometers were sensitive, base noise as low as 2 fT/Hz, and were shown to reduce ambient noise through signal subtraction. When an excitation pulse was introduced, however, residual ringing increased the noise floor; mitigation techniques are discussed. The two detection techniques resulted in comparable Signal-to-Noise Ratio (SNR). Interleaved detection using the atomic magnetometers took half the time of conventional detection and provided localization of the explosives.

Lessons in "Slow" Engagement From Staff and Administrators at a Prebooking Jail Diversion Program.

Objective: In this study, the authors elicited the perspectives of criminal justice and mental health stakeholders about a prebooking jail diversion program, the Judge Ed Emmett Mental Health Diversion Center, serving primarily individuals experiencing chronic homelessness and diagnosed as having a serious mental illness. Methods: The authors analyzed semistructured interviews with 19 participants and observational fieldnotes from 60 hours of ethnographic fieldwork, conducted from January to July 2020 and including five administrative-level meetings. They used qualitative coding to develop themes. Administrative data were also reviewed. Results: Engagement of clients in the program was a major theme. Barriers to engagement included clients' fear of police involvement and strict rules around smoking. Facilitators to engagement included "slow" engagement, or gradual, gentle microengagements over time and across multiple visits, ideally with peer counselors. Conclusions: To promote client use of services at this critical point of care, jail diversion programs might consider ongoing negotiations with clients to balance expectations between the criminal justice and mental health systems of care by using "slow" client engagement, limiting police involvement, and adopting trauma-informed and harm-reduction approaches.

Stem-like breast cancer cells in the activated state resist genetic stress via TGFBI-ZEB1.

Breast cancer cells with stem-like properties are critical for tumor progression, yet much about these cells remains unknown. Here, we characterize a population of stem-like breast cancer cells expressing the integrin αvß3 as transcriptionally related to activated stem/basal cells in the normal human mammary gland. An unbiased functional screen of genes unique to these cells identified the matrix protein TGFBI (BIG-H3) and the transcription factor ZEB1 as necessary for tumorsphere formation. Surprisingly, these genes were not required for cell proliferation or survival, but instead maintained chromosomal stability. Consistent with this finding, CRISPR deletion of either gene synergized with PARP inhibition to deplete αvß3+ stem-like cells, which are normally resistant to this therapy. Our findings highlight a critical role for TGFBI-ZEB1 protection against genetic stress as a key attribute of activated stem-like cells and suggest that disrupting this ability may enhance their "BRCAness" by increasing sensitivity to PARP inhibitors.

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning.

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.

Homogeneous fields: Double expansion method, 3D printing/CNC realization, and verification by atomic magnetometry.

In low-field magnetic resonance applications there is often an interest in creating homogeneous magnetic fields over unusual geometries, particularly when quantum magnetometers are involved. In this paper a design method is proposed, where both the surface current and magnetic field are expanded to find current coefficients that cancel out higher order field terms. Two coils are designed using this double expansion methodology: (1) a tuning field for a half-meter-long atomic magnetometer array and (2) a null field for a magnetometer to operate adjacent to an excitation solenoid. The field verification of the former shows the accuracy of CNC milling and the method proposed; a close analysis of the field signature in the latter revealed the limitations of 3D printing for precise scientific applications. Both coils are designed to be fifth-order error systems or better.

RalGTPases contribute to Schwann cell repair after nerve injury via regulation of process formation.

RalA and RalB are small GTPases that are involved in cell migration and membrane dynamics. We used transgenic mice in which one or both GTPases were genetically ablated to investigate the role of RalGTPases in the Schwann cell (SC) response to nerve injury and repair. RalGTPases were dispensable for SC function in the naive uninjured state. Ablation of both RalA and RalB (but not individually) in SCs resulted in impaired axon remyelination and target reinnervation following nerve injury, which resulted in slowed recovery of motor function. Ral GTPases were localized to the leading lamellipodia in SCs and were required for the formation and extension of both axial and radial processes of SCs. These effects were dependent on interaction with the exocyst complex and impacted on the rate of SC migration and myelination. Our results show that RalGTPases are required for efficient nerve repair by regulating SC process formation, migration, and myelination, therefore uncovering a novel role for these GTPases.

RF atomic magnetometer array with over 40 dB interference suppression using electron spin resonance.

An unshielded array of 87Rb atomic magnetometers, operating close to 1 MHz, is used to attenuate interference by 42-48 dB. A sensitivity of 15 fT/Hz to a local source of signal is retained. In addition, a 2D spectroscopic technique, in which the magnetometers are repeatedly pumped and data acquired between pump times, enables a synchronously generated signal to be distinguished from an interfering signal very close in frequency; the timing and signal mimics what would be observed in a magnetic resonance echo train. Combining the interference rejection and the 2D spectroscopy techniques, a 100 fT local signal is differentiated from a 20 pT interference signal operating only 1 Hz away. A phase-encoded reference signal is used to calibrate the magnetometers in real time in the presence of interference. Key to the strong interference rejection is the accurate calibration of the reference signal across the array, obtained through electron spin resonance measurements. This calibration is found to be sensitive to atomic polarization, RF pulse duration, and direction of the excitation. The experimental parameters required for an accurate and robust calibration are discussed.

Cross-species functional analyses reveal shared and separate roles for Sox11 in frog primary neurogenesis and mouse cortical neuronal differentiation.

A well-functioning brain requires production of the correct number and types of cells during development; cascades of transcription factors are essential for cellular coordination. Sox proteins are transcription factors that affect various processes in the development of the nervous system. Sox11, a member of the SoxC family, is expressed in differentiated neurons and supports neuronal differentiation in several systems. To understand how generalizable the actions of Sox11 are across phylogeny, its function in the development of the frog nervous system and the mouse cerebral cortex were compared. Expression of Sox11 is largely conserved between these species; in the developing frog, Sox11 is expressed in the neural plate, neural tube and throughout the segmented brain, while in the mouse cerebral cortex, Sox11 is expressed in differentiated zones, including the preplate, subplate, marginal zone and cortical plate. In both frog and mouse, data demonstrate that Sox11 supports a role in promoting neuronal differentiation, with Sox11-positive cells expressing pan-neural markers and becoming morphologically complex. However, frog and mouse Sox11 cannot substitute for one another; a functional difference likely reflected in sequence divergence. Thus, Sox11 appears to act similarly in subserving neuronal differentiation but is species-specific in frog neural development and mouse corticogenesis.

Orchestration of Neuronal Differentiation and Progenitor Pool Expansion in the Developing Cortex by SoxC Genes.

As the cerebral cortex forms, specialized molecular cascades direct the expansion of progenitor pools, the differentiation of neurons, or the maturation of discrete neuronal subtypes, together ensuring that the correct amounts and classes of neurons are generated. In several neural systems, the SoxC transcriptional regulators, particularly Sox11 and Sox4, have been characterized as functioning exclusively and redundantly in promoting neuronal differentiation. Using the mouse cerebral cortex as a model, Sox11 and Sox4 were examined in the formation of the most complex part of the mammalian brain. Anticipated prodifferentiation roles were observed. Distinct expression patterns and mutant phenotypes, however, reveal that Sox11 and Sox4 are not redundant in the cortex, but rather act in overlapping and discrete populations of neurons. In particular, Sox11 acts in early-born neurons; binding to its partner protein, Neurogenin1, leads to selective targeting and transactivation of a downstream gene, NeuroD1. In addition to neuronal expression, Sox4 was unexpectedly expressed in intermediate progenitor cells, the transit amplifying cell of the cerebral cortex. Sox4 mutant analyses reveal a requirement for Sox4 in IPC specification and maintenance. In intermediate progenitors, Sox4 partners with the proneural gene Neurogenin2 to activate Tbrain2 and then with Tbrain2 to maintain this cell fate. This work reveals an intricately structured molecular architecture for SoxC molecules, with Sox11 acting in a select set of cortical neurons and Sox4 playing an unanticipated role in designating secondary progenitors.

Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers.

Transcription factors control the fate of a cell by regulating the expression of genes and regulatory networks. Recent successes in inducing pluripotency in terminally differentiated cells as well as directing differentiation with natural transcription factors has lent credence to the efforts that aim to direct cell fate with rationally designed transcription factors. Because DNA-binding factors are modular in design, they can be engineered to target specific genomic sequences and perform pre-programmed regulatory functions upon binding. Such precision-tailored factors can serve as molecular tools to reprogramme or differentiate cells in a targeted manner. Using different types of engineered DNA binders, both regulatory transcriptional controls of gene networks, as well as permanent alteration of genomic content, can be implemented to study cell fate decisions. In the present review, we describe the current state of the art in artificial transcription factor design and the exciting prospect of employing artificial DNA-binding factors to manipulate the transcriptional networks as well as epigenetic landscapes that govern cell fate.

Chemically defined conditions for human iPSC derivation and culture.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

Characterization of systemic hypertension in the San Francisco Chinese community.

This report examines the prevalence of hypertension, its management and control, and the use of antihypertensive medication, diet, and exercise in Chinese adults residing in the San Francisco community. Blood pressure (BP) was measured objectively using an automated oscillometric Dinamap recorder on 708 Chinese adults (295 men and 413 women; age range from 19 to 98 years, mean 59.7), and hypertension, defined as BP >140/90 mm Hg and/or the use of antihypertensive medications, was found in 489 (69%), most of them immigrants from China. Although 202 patients (41%) received antihypertensive medications, only 28 (14%) achieved BP control (<140/90 mm Hg), and in examining the self-management of hypertension, it was found that only 45% of patients used low-sodium diets, and 49% performed regular exercises for > or = 30 minutes > or = 3 times weekly.

Chaperone activity of cytosolic small heat shock proteins from wheat.

Small Hsps (sHsps) and the structurally related eye lens alpha-crystallins are ubiquitous stress proteins that exhibit ATP-independent molecular chaperone activity. We studied the chaperone activity of dodecameric wheat TaHsp16.9C-I, a class I cytosolic sHsp from plants and the only eukaryotic sHsp for which a high resolution structure is available, along with the related wheat protein TaHsp17.8C-II, which represents the evolutionarily distinct class II plant cytosolic sHsps. Despite the available structural information on TaHsp16.9C-I, there is minimal data on its chaperone activity, and likewise, data on activity of the class II proteins is very limited. We prepared purified, recombinant TaHsp16.9C-I and TaHsp17.8C-II and find that the class II protein comprises a smaller oligomer than the dodecameric TaHsp16.9C-I, suggesting class II proteins have a distinct mode of oligomer assembly as compared to the class I proteins. Using malate dehydrogenase as a substrate, TaHsp16.9C-I was shown to be a more effective chaperone than TaHsp17.8C-II in preventing heat-induced malate dehydrogenase aggregation. As observed by EM, morphology of sHsp/substrate complexes depended on the sHsp used and on the ratio of sHsp to substrate. Surprisingly, heat-denaturing firefly luciferase did not interact significantly with TaHsp16.9C-I, although it was fully protected by TaHsp17.8C-II. In total the data indicate sHsps show substrate specificity and suggest that N-terminal residues contribute to substrate interactions.

The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates that these chaperones protect a wide range of cellular functions.

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and co-chaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow.

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate beta(2) integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of approximately 75 pm IL-8, corresponding to ligation of only approximately 10-100 receptors, was sufficient to activate approximately 20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.