Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Polyunsaturated fatty acid analogs act antiarrhythmically on the cardiac IKs channel

Polyunsaturated fatty acids (PUFAs) affect cardiac excitability. Kv7.1 and the β-subunit KCNE1 form the cardiac I_Ks channel that is central for cardiac repolarization. In this study, we explore the prospects of PUFAs as I_Ks channel modulators. We report that PUFAs open Kv7.1 via an electrostatic mechanism. Both the polyunsaturated acyl tail and the negatively charged carboxyl head group are required for PUFAs to open Kv7.1. We further show that KCNE1 coexpression abolishes the PUFA effect on Kv7.1 by promoting PUFA protonation. PUFA analogs with a decreased pK_a value, to preserve their negative charge at neutral pH, restore the sensitivity to open I_Ks channels. PUFA analogs with a positively charged head group inhibit I_Ks channels. These different PUFA analogs could be developed into drugs to treat cardiac arrhythmias. In support of this possibility, we show that PUFA analogs act antiarrhythmically in embryonic rat cardiomyocytes and in isolated perfused hearts from guinea pig.The cardiac action potential is initiated and maintained by inward sodium and calcium currents and terminated by outward potassium currents (1). The I_Ks channel, formed by four α subunits (voltage-gated potassium channel subunit Kv7.1, originally called KCNQ1 or KvLQT1) and two to four auxiliary β subunits (Kv channel beta subunit KCNE1, originally called minK) (1, 2), contributes a major component of the repolarizing potassium current. More than 300 mutations in the genes encoding Kv7.1 and KCNE1 have been identified in patients with cardiac arrhythmia (1). Loss-of-function mutations of the I_Ks channel prolong the QT interval as observed in long QT syndrome, leading to ventricular arrhythmias, ventricular fibrillation, and sudden death (1). Gain-of-function mutations of the I_Ks channel shorten the QT interval, possibly leading to arrhythmia such as short QT syndrome or atrial fibrillation (1). Pharmacological augmentation (in the case of long QT syndrome) or inhibition (in the case of short QT syndrome) of I_Ks channel activity is a logical pharmacological strategy to treat these forms of cardiac arrhythmias.Kv7.1 is a tetrameric voltage-gated K (Kv) channel with six transmembrane segments (called S1–S6) per subunit (3). S5 and S6 from all four subunits together form the pore domain with the central ion-conducting pore. In Kv channels, S6 has been shown to function as the activation gate, shutting off the intracellular access to the pore for K⁺ ions in the closed state of the channel (3–5). Most reported activators or inhibitors of Kv7.1 channels target the ion-conducting pore domain of the channel, opening or blocking the ionic pathway (6–10). S1–S4 of each subunit form a voltage-sensor domain (VSD). In Kv channels, each S4 segment has several positively charged residues and has been shown to move in response to changes in the transmembrane voltage (3, 11). In response to membrane depolarization, the S4 segments move outward with respect to the membrane, which causes channel opening. Although four Kv7.1 subunits per se form a functional channel, Kv7.1 needs to coassemble with the auxiliary β-subunit KCNE1 to recapitulate the biophysical properties of the native cardiac I_Ks channel (12, 13). KCNE1, a single transmembrane helix protein, has been proposed to associate with Kv7.1 in the lipid cleft between adjacent VSDs, making contact with VSD transmembrane segments S1 and S4 and pore transmembrane segment S6 (14–16).In this study, we explore the prospects of polyunsaturated fatty acids (PUFAs) and PUFA analogs as small molecules enhancing or inhibiting the activity of the cardiac I_Ks channel by changing I_Ks channel voltage dependence. We previously suggested that PUFAs facilitate opening of the related Shaker Kv channel via electrostatic attraction of S4 (17–20). The pharmacological sensitivity of I_Ks to small-molecule activators has been shown to depend on the Kv7.1:KCNE1 stoichiometry (21–23). We therefore also determine the impact of Kv7.1:KCNE1 stoichiometry on PUFA sensitivity.Below we show that PUFAs affect the Kv7.1 channel by an electrostatic effect on the voltage sensor movement. We also show that KCNE1 abolishes the PUFA sensitivity of the Kv7.1 channel at physiological pH, suggesting that physiologically occurring PUFAs do not act on I_Ks channels in vivo. Furthermore, we identify PUFA analogs that have effects on the I_Ks channel at physiological pH, increase I_Ks in cardiomyocytes, restore rhythmic firing in arrhythmic cardiomyocytes, and shorten the QT interval in isolated perfused guinea pig hearts. These results may form the basis for development of pharmacological drugs that target the I_Ks channel to prevent cardiac arrhythmias.     

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K⁺ currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2–250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein–coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein–coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.Increased arterial constriction and lack of responsiveness to endogenous vasodilators is a hallmark of vascular disease leading to poor health prognosis. Defining the factors that determine vascular smooth muscle (VSM) activity and modulation by vasorelaxant molecules is therefore imperative for a better understanding of vascular disease. Potassium channels are key regulators of VSM tone because they promote membrane hyperpolarization that limits the activity of voltage-dependent calcium channels known to precipitate vasoconstriction (1). The Kv7 family of voltage-dependent potassium channels and the Kv7.4 isoform, in particular, has a fundamental role in this process. There are five Kv7 isoforms (Kv7.1–Kv7.5) of which Kv7.1, Kv7.4, and Kv7.5 are consistently expressed within VSM, where the predominant molecular architecture is a Kv7.4/Kv7.5 heterotetramer (2, 3). Activation of Kv7 channels produces relaxation of numerous arteries (4–8), whereas blockade of Kv7 channels results in contraction of vessels at rest (7, 9–11) or an inhibition of endogenously derived vasorelaxations (2, 11–13). In addition, molecular reduction of Kv7.4 reduces responses to various Gs-coupled vasodilators in a number of arteries (2, 11). Crucially, Kv7.4 abundance is reduced in various arteries from hypertensive animals (6, 11, 12) where relaxant responses to endogenous vasodilators are also impaired (11, 12). Despite the key role of Kv7.4 channels in the regulation of VSM, and their involvement in mediating Gs-coupled vasodilator responses, the factors that regulate channel activity are poorly understood, and the signals linking Kv7.4 to Gs-receptor activation remain to be elucidated.G-protein–coupled receptor (GPCR) activation promotes the exchange of GDP for GTP resulting in disassociation of the heterotrimeric Gαβγ complex from the receptor into Gα-GTP and Gβγ (14). It is now established that the Gβγ complex as well as the Gα–GTP activates various intracellular signaling pathways (see refs. 15, 16 for reviews). Gβγ subunits also modulate various ion channels directly, a phenomenon of which there are only a handful of examples, with the positive regulation of an inwardly rectifying K⁺ channel in the heart the best characterized (17, 18). In this study, we explored whether Gβγ subunits modulated Kv7.4 channels and therefore function as signaling intermediates following receptor stimulation. Our results show that not only are Gβγ subunits able to enhance Kv7 channels, but also that they are a crucial requirement for the basal activity of the Kv7.4 channel.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

Stress and telomere shortening among central Indian conservation refugees

Research links psychosocial stress to premature telomere shortening and accelerated human aging; however, this association has only been demonstrated in so-called “WEIRD” societies (Western, educated, industrialized, rich, and democratic), where stress is typically lower and life expectancies longer. By contrast, we examine stress and telomere shortening in a non-Western setting among a highly stressed population with overall lower life expectancies: poor indigenous people—the Sahariya—who were displaced (between 1998 and 2002) from their ancestral homes in a central Indian wildlife sanctuary. In this setting, we examined adult populations in two representative villages, one relocated to accommodate the introduction of Asiatic lions into the sanctuary (n = 24 individuals), and the other newly isolated in the sanctuary buffer zone after their previous neighbors were moved (n = 22). Our research strategy combined physical stress measures via the salivary analytes cortisol and α-amylase with self-assessments of psychosomatic stress, ethnographic observations, and telomere length assessment [telomere–fluorescence in situ hybridization (TEL-FISH) coupled with 3D imaging of buccal cell nuclei], providing high-resolution data amenable to multilevel statistical analysis. Consistent with expectations, we found significant associations between each of our stress measures—the two salivary analytes and the psychosomatic symptom survey—and telomere length, after adjusting for relevant behavioral, health, and demographic traits. As the first study (to our knowledge) to link stress to telomere length in a non-WEIRD population, our research strengthens the case for stress-induced telomere shortening as a pancultural biomarker of compromised health and aging.Psychosocial stress is associated with elevated risk for a range of human diseases and curtailment of human life expectancy (1–16). Telomeres—repetitive and stabilizing features of chromosomal termini that cap and protect them—have also been shown to be associated with aging and disease (17–19). Telomere length erodes normally with cell division and generally with aging, triggering cellular senescence once telomere length eclipses a threshold, contributing to tissue degeneration and organ decline with longevity (20–25). Given evidence that stress can elevate the risk of human mortality, and that premature telomere shortening can serve as a proxy for increased risk of disease and mortality, it is reasonable to posit that stress is also associated with telomere shortening (23, 26, 27). Indeed, research provides evidence of telomere shortening in a range of stress-inducing life situations, including among primary caregivers of chronically ill children and Alzheimer’s patients (28, 29), children spending more time in orphanages or experiencing other forms of neglect and adversity (30–32), women suffering from intimate partner violence (33), patients suffering from stress-related mood disorders (34, 35), and individuals of low socioeconomic status (SES) (36). [However, not all research demonstrates the expected associations between stress and telomere length, as in studies failing to identify links between low SES and telomere shortening (27, 37).]Of note, studies relating stress and telomere maintenance typically unfold in what have been termed “WEIRD” societies (38, 39)—i.e., within Western, educated, industrialized, rich, and democratic populations, a small slice of total humanity—potentially limiting our understanding and generalizability of the association between biopsychosocial stressors and telomere maintenance. Populations in WEIRD societies generally enjoy higher life expectancies (40–42) and are relatively insulated from the traumas, stressors, and political coercions common throughout the developing world. Insofar as telomere length proxies for life expectancy, and that psychosocial well-being increases with economic development (43), studies involving WEIRD populations can be construed as potentially sampling high on the telomere length dimension and low on the stress dimension.By contrast, we assessed associations between stress and telomere length in the context of tumultuous life changes experienced by an indigenous population—the Sahariya—displaced from their ancestral homes in a central Indian wildlife sanctuary. There, we examined adult heads of household in two representative villages, one relocated from their forest homes in the ecological core of the sanctuary to accommodate the future introduction of Asiatic lions into the sanctuary, and the other newly isolated in the sanctuary’s buffer zone after their previous neighbors were relocated, with this second group of villagers also facing more restricted forest access in this buffer zone (44). Consistent with prior research on the lasting psychosomatic costs of displacement, dispossession, and loss of homeland, a phenomenon found mainly in the developing world, our sample of villagers presented with levels of psychosomatic suffering that approximate populations receiving psychiatric care (45, 46). Developing and underdeveloped societies are estimated to host 80% of the estimated count of refugees and displaced peoples worldwide (46). Indeed, adult Sahariya villagers expressed deep uncertainty about the future, particularly with respect to the welfare of their children, and wished to return to their predisplacement lives.Although displacement of human populations is an intrinsically compelling phenomenon, with an estimated 51.2 million internally displaced peoples worldwide (46, 47), our investigation of the relationship between stress and telomere length in a non-WEIRD setting is motivated in this context by other scientific reasons. Specifically, our population of indigenous Sahariya potentially demonstrates compromised telomere maintenance (given measurably lower life expectancies in rural parts of central India), while being higher on the stress dimension (given the high levels of reported psychosomatic suffering and disease burden), providing valuable information on a unique and understudied population, as well as advancing efforts to more fully characterize statistical associations between stress and telomere maintenance.^†It is also important to recognize that such real-life studies are often characterized by methodological limitations. For example, research linking life stress and telomere shortening typically rely on psychosocial distress scales or comparisons between presumed or perceived stressed and nonstressed control groups, rather than physical measures such as salivary stress analytes (like cortisol): e.g., orphans vs. nonorphans, abused vs. nonabused women, low- vs. high-SES individuals, and the mentally disordered vs. mentally healthy (19, 28, 30–35, 55). In vitro research demonstrates that the stress hormone cortisol can interfere with maintenance of telomere length by reducing telomerase activity (56). It has also been shown that oxidative stress preferentially damages telomeric DNA compared with other genomic regions, and that antioxidants can delay “replicative senescence” (57–59). However, in vivo research linking self-reported levels of experienced psychosocial stress, stress biomarkers (i.e., catecholamines and glucocorticoids), and telomere maintenance are few (60, 61), severely limiting our understanding of potential connections and hypothesized mechanisms, such as inflammation and oxidative stress (60, 62), in naturally occurring contexts. Furthermore, current population-based stress and telomere studies typically rely on quantitative real-time PCR methodology, which evaluates all of a cell’s DNA to assess an average telomere length across many different cell types (19, 28, 63). Although certainly representing progress in the field, such an approach is still limited by lack of specificity, not only in regard to cell type, but also in evaluation of particular populations of telomeres (e.g., distributions of shortest and/or longest).In contrast to previous studies, here we combined physical measures of the psychobiology of the stress response (salivary cortisol and α-amylase), collected in these Indian village contexts using minimally invasive field-appropriate techniques (64–72), with Sahariya self-assessments of psychosomatic stress, ethnographic observations, and high-resolution telomere length measurement. The value of microscopy-based techniques for evaluation of telomere length on a cell-by-cell basis has been effectively demonstrated (73–77). We developed an innovative approach for assessment of telomere length that combined telomere–fluorescence in situ hybridization (TEL-FISH) (73) using a modified protocol to collect and process the obtained samples (78), with 3D reconstruction of individual cell nuclei to facilitate analysis of all visible telomere signals in the entire extension of sampled cells, rather than just a fraction (presumably the longest) of them (75). Our approach allowed us to evaluate the length of hundreds of thousands of individual telomeres in a single class of putative buccal basal stem or progenitor cells, greatly improving the specificity and quantification of telomere length, including the important ability to define distributions of the shortest telomeres (76, 77). Such a strategy afforded us particularly high-resolution data amenable to multilevel statistical analysis.To summarize, with innovative methodologies and strategies that mapped community and individual variation in life stress in a unique non-Western setting, our research explores the case for stress-related telomere shortening as a pancultural biomarker of compromised health and aging.     

Stress controls the mechanics of collagen networks

Collagen is the main structural and load-bearing element of various connective tissues, where it forms the extracellular matrix that supports cells. It has long been known that collagenous tissues exhibit a highly nonlinear stress–strain relationship, although the origins of this nonlinearity remain unknown. Here, we show that the nonlinear stiffening of reconstituted type I collagen networks is controlled by the applied stress and that the network stiffness becomes surprisingly insensitive to network concentration. We demonstrate how a simple model for networks of elastic fibers can quantitatively account for the mechanics of reconstituted collagen networks. Our model points to the important role of normal stresses in determining the nonlinear shear elastic response, which can explain the approximate exponential relationship between stress and strain reported for collagenous tissues. This further suggests principles for the design of synthetic fiber networks with collagen-like properties, as well as a mechanism for the control of the mechanics of such networks.Collagen type I is the most abundant protein in mammals where it serves as the primary component of many load-bearing tissues, including skin, ligaments, tendons, and bone. Networks of collagen type I fibers exhibit mechanical properties that are unmatched by manmade materials. A hallmark of collagen and collagenous tissues is a dramatic increase in stiffness when strained. Qualitatively, this property of strain stiffening is shared by many other biopolymers, including intracellular cytoskeletal networks of actin and intermediate filaments (1–5). On closer inspection, however, collagen stands out from the rest: it has been shown that collagenous tissues exhibit a regime in which the stress is approximately exponential in the applied strain (6). The origins of this nonlinearity are still not known (7, 8), and existing models for biopolymer networks cannot account quantitatively for collagen. In particular, it is unknown whether the nonlinear mechanical response of collagen originates at the level of the individual fibers (1, 3, 9, 10) or arises from nonaffine network deformations as suggested by numerical simulations (11–17).Here, we present both experimental results on reconstituted collagen networks, as well as a model that quantitatively captures the observed nonlinear mechanics. Our model is a minimal one, of random networks of elastic fibers possessing only bending and stretching elasticity. This model can account for our striking experimental observation that the stiffness of collagen becomes independent of protein concentration in the nonlinear elastic regime, over a range of concentrations and applied shear stress. Our model highlights the importance of local network geometry in determining the strain threshold for the onset of nonlinear mechanics, which can account for the concentration independence of this threshold that is observed for collagen (8, 17), in strong contrast to other biopolymer networks. Finally, our model points to the important role of normal stresses in determining the nonlinear shear elastic response, including the approximate exponential relationship between stress and strain reported for collagenous tissues (6).     

Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents

A simultaneous increase in cytosolic Zn²⁺ and Ca²⁺ accompanies the initiation of neuronal cell death signaling cascades. However, the molecular convergence points of cellular processes activated by these cations are poorly understood. Here, we show that Ca²⁺-dependent activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is required for a cell death-enabling process previously shown to also depend on Zn²⁺. We have reported that oxidant-induced intraneuronal Zn²⁺ liberation triggers a syntaxin-dependent incorporation of Kv2.1 voltage-gated potassium channels into the plasma membrane. This channel insertion can be detected as a marked enhancement of delayed rectifier K⁺ currents in voltage clamp measurements observed at least 3 h following a short exposure to an apoptogenic stimulus. This current increase is the process responsible for the cytoplasmic loss of K⁺ that enables protease and nuclease activation during apoptosis. In the present study, we demonstrate that an oxidative stimulus also promotes intracellular Ca²⁺ release and activation of CaMKII, which, in turn, modulates the ability of syntaxin to interact with Kv2.1. Pharmacological or molecular inhibition of CaMKII prevents the K⁺ current enhancement observed following oxidative injury and, importantly, significantly increases neuronal viability. These findings reveal a previously unrecognized cooperative convergence of Ca²⁺- and Zn²⁺-mediated injurious signaling pathways, providing a potentially unique target for therapeutic intervention in neurodegenerative conditions associated with oxidative stress.Calcium has long been recognized as a critical component of neuronal cell death pathways triggered by oxidative, ischemic, and other forms of injury (1). Indeed, Ca²⁺ deregulation has been associated with a variety of detrimental processes in neurons, including mitochondrial dysfunction (2), generation of reactive oxygen species (3), and activation of apoptotic signaling cascades (4). More recently, zinc, a metal crucial for proper cellular functioning (5), has been found to be closely linked to many of the injurious conditions in which Ca²⁺ had been thought to play a prominent role (6–10). In fact, it has been suggested that a number of deleterious properties initially attributed to Ca²⁺ may have significant Zn²⁺-mediated components (11, 12). Although it is virtually impossible to chelate, or remove, Ca²⁺ without disrupting Zn²⁺ levels (13), the introduction of techniques to monitor Ca²⁺ and Zn²⁺ simultaneously in cells (14) has made it increasingly apparent that both cations have important yet possibly distinct roles in neuronal cell death (12, 15–18). However, the relationship between the cell death signaling pathways activated by the cations is unclear, and possible molecular points of convergence between these signaling cascades have yet to be identified.Injurious oxidative and nitrosative stimuli lead to the liberation of intracellular Zn²⁺ from metal binding proteins (19). The released Zn²⁺, in turn, triggers p38 MAPK- and Src-dependent Kv2.1 channel insertion into the plasma membrane, resulting in a prominent increase in delayed rectifier K⁺ currents in dying neurons, with no change in activation voltage, ∼3 h following a brief exposure to the stimulus (20–26). The increase in Kv2.1 channels present in the membrane mediates a pronounced loss of intracellular K⁺, likely accompanied by Cl⁻ (27, 28), that facilitates apoptosome assembly and caspase activation (20, 29–34). Indeed, K⁺ efflux appears to be a requisite event for the completion of many apoptotic programs, including oxidant-induced, Zn²⁺-mediated neuronal death (21).Ca²⁺ has been suggested to regulate the p38 MAPK signaling cascade via Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-mediated activation of the MAP3K apoptosis signaling kinase-1 (ASK-1) (35). Because ASK-1 is also required for p38-dependent manifestation of the Zn²⁺-triggered, Kv2.1-mediated enhancement of K⁺ currents (36), we hypothesized that the p38 activation cascade may provide a point of convergence between Ca²⁺ and Zn²⁺ signals following oxidative injury. Here, we report that Ca²⁺ and Zn²⁺ signals do, in fact, converge on a cellular event critical for the K⁺ current enhancement, and that CaMKII is required for this process. However, CaMKII does not act upstream of p38 activation as originally hypothesized, but instead interacts with the N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein syntaxin, which we showed to be necessary for the insertion of Kv2.1-encoded K⁺ channels following an apoptotic stimulus (23).     

Selective Kv1.3 channel blocker as therapeutic for obesity and insulin resistance

Obesity is an epidemic, calling for innovative and reliable pharmacological strategies. Here, we show that ShK-186, a selective and potent blocker of the voltage-gated Kv1.3 channel, counteracts the negative effects of increased caloric intake in mice fed a diet rich in fat and fructose. ShK-186 reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. These changes mimic the effects of Kv1.3 gene deletion. ShK-186 did not alter weight gain in mice on a chow diet, suggesting that the obesity-inducing diet enhances sensitivity to Kv1.3 blockade. Several mechanisms may contribute to the therapeutic benefits of ShK-186. ShK-186 therapy activated brown adipose tissue as evidenced by a doubling of glucose uptake, and increased β-oxidation of fatty acids, glycolysis, fatty acid synthesis, and uncoupling protein 1 expression. Activation of brown adipose tissue manifested as augmented oxygen consumption and energy expenditure, with no change in caloric intake, locomotor activity, or thyroid hormone levels. The obesity diet induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. This action on the liver may underlie the differential effectiveness of ShK-186 in mice fed a chow vs. an obesity diet. Our results highlight the potential use of Kv1.3 blockers for the treatment of obesity and insulin resistance.Obesity and its related metabolic disorders are a global pandemic. In the United States, ∼65% of the population is either overweight or obese (1). The complications of obesity will impose a significant burden on health care systems the world over (2, 3). Medical therapeutic options are limited. Therapies with novel mechanisms of action to combat obesity would therefore have significant medical and economic impact.Kv1.3, a voltage-gated K⁺ channel (K_v), has been reported to regulate energy homeostasis and body weight (4). Importantly, Kv1.3^−/− mice fed a high-fat diet exhibit increased light-phase metabolism together with reduced weight gain; lower levels of blood sugar, leptin, and insulin; and increased energy expenditure in comparison with wild-type littermates (4–10) (Table S1). Similarly, in melanocortin receptor 4 knockout mice, a genetic model of obesity, Kv1.3 gene deletion increases energy expenditure; decreases adiposity and weight; reduces leptin levels; and extends life span (8). Two mechanisms have been proposed to explain the antiobesity and metabolic effects of Kv1.3 gene deletion: enhanced peripheral insulin sensitization and augmented olfaction. First, knockout of the Kv1.3 gene or pharmacological blockade of the channel with the scorpion toxin margatoxin has been reported to enhance peripheral insulin sensitivity in white adipocytes and skeletal muscle by promoting translocation of the glucose transporter GLUT4 to the plasma membrane and thereby increasing glucose uptake (6, 7). Second, the olfactory bulb expresses Kv1.3 and is reported to function as a metabolic sensor (5, 9). Removal of the olfactory bulb attenuates the weight-reducing effects of Kv1.3 gene deletion (9). In humans, a polymorphism in the Kv1.3 promoter is associated with impaired glucose tolerance and with lower insulin sensitivity (11). These results suggest that selective Kv1.3 blockers might have use in the management of obesity and insulin resistance.We used a mouse model of diet-induced obesity and insulin resistance to evaluate the therapeutic effects of a selective Kv1.3 blocker. In this model, mice fed an obesity-inducing diet rich in both fat and fructose (Tables S2 and S3) gain significant weight and adiposity, and develop insulin resistance within 6–8 wk (12–15). As a Kv1.3 blocker we chose ShK-186 because of its picomolar potency (IC₅₀ 69 pM), >100-fold selectivity over closely related channels, weak immunogenicity, and excellent safety profile in rodents and nonhuman primates (16–21). Furthermore, ShK-186 is the first Kv1.3-specific blocker to advance to human safety trials as a potential therapeutic for autoimmune diseases. Our study highlights the powerful antiobesity effects of ShK-186, and we characterize mechanisms that may contribute to ShK-186’s therapeutic activity.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion.Taxis, the directed kinematic response to external signals, is a defining feature of living things that affects their reproduction, foraging, migration, and survival strategies (1–4). Higher organisms rely on sophisticated networks of finely tuned sensory mechanisms to move efficiently in the presence of chemical or physical stimuli. However, various fundamental forms of taxis are already manifest at the unicellular level, ranging from chemotaxis in bacteria (5) and phototaxis in unicellular green algae (2) to the mechanical response (durotaxis) of fibroblasts (6) and rheotaxis (7, 8) in spermatozoa (3, 9–12). Over the last few decades, much progress has been made in deciphering chemotactic, phototactic, and durotactic pathways in prokaryotic and eukaryotic model systems. In contrast, comparatively little is known about the physical mechanisms that enable flow gradient sensing in sperm cells (3, 9–13). Recent studies (3, 12) suggest that mammalian sperm use rheotaxis for long-distance navigation, but it remains unclear how shear flows alter flagellar beat patterns in the vicinity of surfaces and, in particular, how such changes in the beat dynamics affect the steering process. Answering these questions will be essential for evaluating the importance of chemical (14) and physical (4) signals during mammalian fertilization (15–17).A necessary requirement for any form of directed kinematic response is the ability to change the direction of locomotion. Multiflagellate bacteria achieve this feat by varying their motor activity, resulting in alternating phases of entangled and disentangled flagellar dynamics that give rise to run-and-tumble motion (5). A similar mechanism was recently discovered in the biflagellate eukaryote Chlamydomonas reinhardtii (18). This unicellular green alga actively redirects its swimming motion through occasional desynchronization of its two cilia (19), although it is still debated whether this effect is of mechanical (20) or hydrodynamic (21, 22) origin. Experiments (23) show that the alga’s reorientation dynamics can lead to localization in shear flow (24, 25), with potentially profound implications in marine ecology. In contrast to taxis in multiflagellate organisms (2, 5, 18, 26, 27), the navigation strategies of uniflagellate cells are less well understood. For instance, it was discovered only recently that uniflagellate marine bacteria, such as Vibrio alginolyticus and Pseudoalteromonas haloplanktis, use a buckling instability in their lone flagellum to change their swimming direction (28). However, as passive prokaryotic flagella differ fundamentally from their active eukaryotic counterparts, it is unclear to what extent such insights translate to spermatozoa.Earlier studies of human sperm locomotion have identified several potential steering and transport mechanisms, including thermotaxis (4), uterine peristalsis (29, 30), and chemotaxis (14, 16, 31), but their relative importance has yet to be quantified. Recent experiments (3, 32, 33) demonstrate that rheotaxis, combined with steric surface alignment (12, 34), enables robust long-distance navigation by turning sperm cells preferentially against an externally imposed flow direction (9, 10), but how exactly this realignment process happens is unknown. It has been suggested (32, 35, 36) that the intrinsic curvature or chiral beat dynamics (37, 38) of the flagellum could play an essential role in rheotactic steering, but this remains to be confirmed in experiments. Similarly, an increasing number of theoretical models (36, 39–47) still await empirical validation, because 3D data for the beat pattern of sperm swimming close to surfaces has been lacking.To examine the dynamics of human sperm rheotaxis quantitatively, we here combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction. Single-cell tracking reveals the existence of two kinematically distinct swimming states that result in opposite turning behaviors under flow reversal. We quantify this effect for a range of viscosities and shear rates, and use these comprehensive data to constrain an effective 2D model through a systematic large-scale scan ( > 6,000 parameter combinations). To identify the details of the flagellar beat dynamics during rheotaxis, we developed an algorithm that translates 2D intensity profiles into 3D positional data. Our 3D analysis confirms that human sperm perform a rolling motion (48), characterized by weakly nonplanar beat patterns and a rotating beat plane. However, contrary to current beliefs, we find that neither the rolling direction nor beat helicity determine the turning direction after flow reversal. Instead, the rheotactic turning behavior correlates with a previously unrecognized asymmetry in the midpiece, likely caused by a buckling instability. These findings call for a revision and extension of current models (36, 39–44, 46).     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Longitudinal spread of mechanical excitation through tectorial membrane traveling waves

The mammalian inner ear separates sounds by their frequency content, and this separation underlies important properties of human hearing, including our ability to understand speech in noisy environments. Studies of genetic disorders of hearing have demonstrated a link between frequency selectivity and wave properties of the tectorial membrane (TM). To understand these wave properties better, we developed chemical manipulations that systematically and reversibly alter TM stiffness and viscosity. Using microfabricated shear probes, we show that (i) reducing pH reduces TM stiffness with little change in TM viscosity and (ii) adding PEG increases TM viscosity with little change in TM stiffness. By applying these manipulations in measurements of TM waves, we show that TM wave speed is determined primarily by stiffness at low frequencies and by viscosity at high frequencies. Both TM viscosity and stiffness affect the longitudinal spread of mechanical excitation through the TM over a broad range of frequencies. Increasing TM viscosity or decreasing stiffness reduces longitudinal spread of mechanical excitation, thereby coupling a smaller range of best frequencies and sharpening tuning. In contrast, increasing viscous loss or decreasing stiffness would tend to broaden tuning in resonance-based TM models. Thus, TM wave and resonance mechanisms are fundamentally different in the way they control frequency selectivity.The sharp frequency selectivity of auditory nerve fiber responses to sound is a hallmark of mammalian cochlear function. This remarkable signal processing originates in the mechanical stage of the cochlear signal processing chain (1–7), as evidenced by measured motions and mechanical properties of the basilar membrane (BM) (2–9) and tectorial membrane (TM) (10–24). Although the hydromechanical mechanisms underlying BM motions have been characterized based on experimental and theoretical studies, the mechanisms underlying TM motions remain unclear.The TM is an acellular matrix that overlies the hair bundles of sensory receptor cells. Based on its strategic position above the organ of Corti, conventional cochlear models (25–29) have implicated local mechanical properties (i.e., mass, stiffness) of the TM in stimulating the sensory hair bundles of hair cells and in cochlear tuning. Recent dynamic measurements of the TM, in vitro (17, 30–33) and in vivo (34), suggest that the TM supports longitudinal coupling, with large spatial extents across a broad range of frequencies. This longitudinal coupling manifests in the form of propagating traveling waves that are thought to contribute to hearing mechanisms (17, 21, 30, 35–40). Genetic modification studies provide further support that the spatial extent of TM waves may play a significant role in cochlear tuning (30, 32). Although these measurements, models, and genetic modification studies have confirmed the importance of TM mechanical properties in hearing, they have not isolated the distinct roles of TM stiffness and viscosity in generating longitudinally propagating traveling waves of the TM.To understand the contributions of TM material properties on traveling waves better, we developed chemical manipulations to alter the stiffness and viscosity of the TM selectively and reversibly. Because the TM is poroelastic (32, 41), we expect that changes in bath composition can have a direct effect on the mechanical properties of the TM mechanical matrix and its interstitial fluid, which makes up 97% of TM wet weight (42). The addition of PEG has previously been shown to generate an osmotic response that could be accounted for by the permeability of these molecules through the matrix rather than by direct changes to the matrix itself (41). In contrast, changing bath pH has little effect on the osmotic pressure or viscosity of the bath but has been shown to have a direct effect on the macromolecular matrix (43). In this paper, we apply these physicochemical manipulations to alter TM material properties reversibly, and thereby probe their role in controlling longitudinal spread of excitation through the TM.