Neutrophils are short-lived white blood cells derived from bone marrow myeloid precursors. Attention to their potential role in human cancer has largely been ignored. New findings suggest that the role of neutrophils in cancer-related inflammation may need careful reappraisal of infiltration, polarization, and prognostic significance of neutrophils in human cancer [1]. Previous older studies have advocated a role of direct or antibody-dependent killing of tumor cells by this website neutrophils (recently reviewed

in [2]). One of the oldest reports of a potential anti-tumor effect of the innate immune system comes from William Coley (reviewed in [3]). He observed that patients with febrile streptococcal

infection within selleck inhibitor an ulcerated tumor had a better prognosis than patients with uninfected tumors. For obvious reasons, he was unable to explain the observations in immunology terms. Since Coley’s initial reports, knowledge about mechanisms in cellular immunity has increased. However, cancer therapy for invasive human cancer utilizing a neutrophil-mediated approach has not yet been established [4]. This review will focus on recently published articles regarding immunomonitoring of neutrophils in blood and tumor tissue in clinical trials comprising the main human tumor types, with a strong emphasis on independent prognostic relevance assessed by multivariate analyses. The first report of neutrophils as an adverse prognostic factor for patients with metastatic renal cell carcinoma (mRCC) was published in 1996 by Lopez Hanninen et al. [5], Table 1. In a series of 215 consecutive patients with mRCC treated with interleukin-2 (IL-2) based immunotherapy, elevated baseline blood neutrophils (>6 × 109/L) was identified as an independent risk factor of short overall survival (OS). Subsequently, the Groupe Français d‘Immunothérapie

published in 2002 results comprising 782 patients in successive multicenter trials using cytokine regimens [6]. Analyses were performed on this large prospective database to identify prognostic factors for survival and predictive factors for progression. Elevated baseline neutrophil count (>7.5 × 109/L) was independently predictive of short Linifanib (ABT-869) survival in a multivariate analysis. The authors also identified four independent factors predictive of rapid progression under cytokine treatment: presence of hepatic metastases, <1 year from renal tumor to metastases, more than one metastatic site, and elevated baseline neutrophil counts (>7.5 × 109/L). Patients who combined at least three of these factors had >80% probability of rapid progression despite treatment. The authors stated that these results should be taken into account when making the decision to treat with cytokines.

Morphologically, the Ruffini ending is similar to the Golgi tendon organ, it is a large (200–100 μm) and thin spindle-shaped cylinder composed

of layers of perineural tissue including Schwann cells and collagen fibers and an inner core composed of nerve terminals surrounded by a capsule space filled with fluid (Chambers et al., 1972 and Halata, 1977). In humans, each SAII axon possesses a low-threshold region, suggesting that a single Aβ fiber supplies each receptor organ (Johansson and Vallbo, 1980). Unlike the Merkel cell-neurite complex, the Aβ fibers that make up SAII-LTMRs are suggested to sense mechanical stretch applied to collagen fibers of the Ruffini ending (Maeda et al., 1999 and Rahman et al., 2011). It is unlikely, however, that in

the mouse Ruffini endings or Ruffini-like structures give rise to SAII-LTMR responses, as such structures have not been identified in rodents. Furthermore, rodent SAII-LTMRs have been observed Panobinostat supplier after stimulation of hairy skin in an ex vivo skin/nerve preparation in which deep structures such as muscles and associated joints are removed (Wellnitz et al., 2010 and Zimmermann et al., 2009). Therefore, the functions of SAII-LTMRs in different animal species and the morphological properties of SAII-LTMR endings remain unknown. The other physiologically defined mechanosensor is the RA receptor, which responds best to objects moving across the skin but less well to static indentation. As with SA-LTMRs, RA-LTMRs can be further divided into two categories: RAI- and RAII-LTMRs. In the simplest interpretation, Phospholipase D1 they merge into a psychophysical frequency Protein Tyrosine Kinase inhibitor continuum, with RAI responses generally associated with small receptive fields and low-frequency vibrations, such as tapping and flutter (1–10 Hz), while RAII responses are associated with larger receptive fields and high-frequency vibrations (from 80–300 Hz) (Knibestol, 1973, Talbot et al., 1968 and Vallbo and Johansson, 1984). Anatomically, both are associated with corpuscles, which may be significant to both their rapidly adaptive properties and the tactile functions they subserve. RAI-LTMRs and Meissner Corpuscles. One of the hallmarks of rapidly adapting responses

is the firing of action potentials only at the initial and final contacts of a mechanical stimulus (Table 1). The percept initially associated with activation of RAI-LTMRs innervating the hand was the feeling of rapid skin movement or “flutter,” and, therefore, the first function ascribed to RAI-LTMRs was detection and scaling of low-frequency vibrations (Torebjörk and Ochoa, 1980). However, RAI-LTMRs possess other response properties that may be specialized for a unique function in grip control. First, in comparison to SAI-LTMRs, RAI-LTMRs are about four times more sensitive, yet respond with far less spatial acuity to stimuli moving across their receptive fields. Second, RAI-LTMRs respond consistently and with very short latencies to skin stimulation.

, 2008), GSK1120212 mw providing one potential mechanism for stress-induced deficits in memory recall (Chen et al., 2010). Similarly, using transcranial two-photon microscopy to image the dynamic remodeling of postsynaptic dendritic spines in the living, developing cortex (Liston and Gan, 2011), we found that glucocorticoids have rapid effects on both spine formation and elimination within hours of exposure. Surprisingly, low-dose dexamethasone (0.1 mg/kg), a synthetic glucocorticoid that inhibits endogenous corticosteroid synthesis without penetrating the blood/brain barrier

(Karssen et al., 2005), effectively prevented developmental spine formation and pruning. It is important to note that studies in neuronal cultures and in the developing cortex are investigating spine remodeling under conditions of heightened plasticity, so additional work will be needed to understand how the results apply to the adult brain. However, these experiments indicate that glucocorticoids play an unexpected, necessary role in facilitating physiological spine maturation in the developing adolescent brain, acting on timescale of Birinapant in vivo minutes to hours to facilitate spine remodeling. These unexpectedly

rapid effects also suggest that circadian glucocorticoid oscillations may contribute to synaptic plasticity during learning and development. To test this hypothesis, we conducted a series of two-photon imaging studies in mice before and after training on a RotaRod motor Libraries skill-learning paradigm, and found that

circadian glucocorticoid peaks and troughs play critical, complementary roles in facilitating experience-dependent spine remodeling (Fig. 2c–g) (Liston et al., 2013). Specifically, circadian glucocorticoid peaks enhanced spine formation rapidly in the hours after learning, acting through a glucocorticoid receptor-dependent, non-transcriptional mechanism. In accord with prior reports (Yang et al., 2009), training increased formation rates but only if it occurred during the circadian peak. In mice that were trained during the circadian trough, spine formation rates were equivalent to those of Parvulin untrained mice, and memory retention was reduced one week later. Furthermore, circadian troughs were necessary for stabilizing a subset of learning-related spines and pruning a corresponding set of pre-existing synapses. Memory retention and the long-term survival of learning-related spines required intact circadian troughs in the days after learning, which enhanced learning-related spine pruning through a distinct, mineralocorticoid receptor-dependent, transcriptional mechanism. In this way, circadian glucocorticoid oscillations were critical for maintaining homeostasis in synaptic density, by balancing formation and pruning after learning to maintain relatively stable synaptic densities despite repeated bouts of learning-related remodeling.

An additional outstanding issue that should also be addressed in future studies is whether progressive resistance training alone can change physical activity levels. Progressive resistance training is one Libraries possible exercise and recreation option for adolescents with Down syndrome. Previous studies have investigated the effectiveness of other exercise options in this population such as aerobic training and circuit training (Khalili and Elkins 2009, Millar et al 1993, Weber and French 1988). The predominance of males who volunteered to participate in the current study might suggest that it is more socially desirable

for males to take part in progressive resistance training. The prevalence of Down syndrome is approximately 10% higher among males than females (Shin et al 2009), so more males self-selected into this study than would be expected on the basis AZD8055 datasheet of population distribution alone. In conclusion, progressive resistance training led by physiotherapy student

mentors and performed in a community gymnasium is a feasible, socially desirable, and safe exercise option for adolescents with Down syndrome that can lead to improvements in lower-limb muscle selleck products performance. This trial provides important data that justify a future randomised trial to ascertain whether progressive resistance training carries over into an improved ability for adolescents with Down syndrome to complete daily tasks and physical activities. eAddenda: Table 3 available at www.jop.physiotherapy.asn.au Ethics: The trial received ethics approval from the La Trobe University Human Ethics Committee (08–024). Written informed consent to the research was obtained from the parents of all participants. Support: Windermere Foundation. The authors acknowledge the contributions of all the participants and their families. Competing interests: None declared. “
“Post-stroke shoulder pain is a frequent and disabling condition that has been reported in up to 85% of people who attend rehabilitation

(Bender and McKenna 2001, Turner-Stokes and Jackson 2002), and in one-third of stroke survivors in general (Lingdgren et al 2007, Ratnasabapathy et al 2003). Moderate others to severe levels of pain are often reported (Lingdgren et al 2007), which can restrict participation in daily activities and rehabilitation, and degrade quality of life (Bender and McKenna 2001, Chae et al 2007). Many factors are proposed to contribute to poststroke shoulder pain, but these are not well understood. This limits effective management of this disabling condition (Bender and McKenna 2001, Turner-Stokes and Jackson 2002). Clinicians need a thorough understanding of the factors that increase the risk of post-stroke shoulder pain in order to identify patients at risk and implement strategies to prevent and manage this disabling condition (Nicks et al 2007, Turner-Stokes and Jackson 2002).

While the effect of MPEP in the NSF was not attenuated by NBQX in the present study, we reported that the effect of ketamine was blocked by NBQX in the same paradigm. Therefore, the mGlu5 receptor antagonist may increase 5-HT release via a different neural mechanism from that of ketamine, i.e., an AMPA receptor-independent mechanism, which may explain the involvement of distinct 5-HT receptor subtypes Dolutegravir price in the effects in the NSF test. The neural mechanism of 5-HT release and the activation of the 5-HT2A/2C receptor induced by an mGlu5 receptor

antagonist in the NSF test remain to be elucidated. Treatment with MTEP reportedly increases 5-HT release without elevating 5-HTIAA in the prefrontal cortex in rats, indicating that the blockade of the mGlu5 receptor may inhibit the 5-HT transporter to increase 5-HT release (21).

However, Heidbreder et al. (2003) reported that MPEP had a moderate affinity for the norepinephrine (NE) transporter, but not for the 5-HT transporter, as evaluated using radioligand binding assays (26). Moreover, 5-HT transporter inhibitors reportedly do not exert an effect after acute treatment selleck chemicals llc in the NSF test (28), which is in accord with our previous finding (22). Therefore, it is unlikely that an mGlu5 receptor antagonist increases 5-HT release by inhibiting the 5-HT transporter. Of note, a previous study showed that gene deletion of the mGlu5 receptor in mice increased the behavioral response to a 5-HT2A receptor agonist, suggesting PD184352 (CI-1040) that blockade of the mGlu5 receptor may enhance the sensitivity to the 5-HT2A receptor (29). Moreover, 5-HT2 receptors are positioned on GABAergic neurons (30), and the stimulation of 5-HT2 receptors increases GABA release in the prefrontal cortex (31). Given that the GABAergic system is known to be disrupted in depressed patients (for a review, see Ref. (32)), it is intriguing to speculate that regulation of the GABAergic system

via the 5-HT2 receptor may be involved in the antidepressant effect of mGlu5 receptor antagonists. The present study has a notable limitation. The specificity of the mGlu5 receptor antagonist, MPEP was not optimal, as it also inhibits the NMDA receptor and NE transporter (26) and (33) as well as acting as a positive allosteric modulator of the mGlu4 receptor (34). However, MPEP acts on the above-mentioned receptors and transporter at a concentration more than 1000 times higher than that blocks the mGlu5 receptor (an IC50 value of 36 nM) (35), and MPEP did not exhibit an antidepressant-like effect in mGlu5 receptor-knockout mice in the forced swimming test (36). Thus, the effect of MPEP at a dose 3 mg/kg can most likely be attributed to the blockade of the mGlu5 receptor.

However clear negative and positive themes emerged suggesting this was not the case. Clinicians had both positive and negative perceptions about their involvement in a clinical trial. However, there was a consensus that all of the clinicians were interested in participating in future research, suggesting RO4929097 cell line that the positive experiences outweighed the negative. In the future, evidencebased practice will only be possible if clinicians

participate in clinical trials and adhere to the protocols so that an accurate evidence base is built up. A trial that fits into the way physiotherapy departments deliver their service should be more acceptable to both therapists and administrators. The features that make a trial more appealing – such as a clinically feasible and relevant intervention, support from a dedicated research team, and provision of equipment to make the delivery of the intervention Libraries efficient – if incorporated in to the design of future trials, may increase clinical commitment to research. Ethics: Approval for this study was granted by the Human Research Ethics Committee

of The University of Sydney (08-2002/2916). All participants provided written consent. Competing interests: Nil Support: KRX-0401 molecular weight University of Sydney sesquicentenary grant; NHMRC (Australia) project grant (402679). We are grateful to the physiotherapists who delivered the intervention and particularly those who gave up their time to be interviewed. “
“During rehabilitation, inpatients spend relatively little time

receiving therapy (Bernhardt et al 2004, Thompson and until McKinstry 2009). Additional physiotherapy reduces length of stay and improves mobility, activity, and quality of life for people in acute and rehabilitation settings (Peiris et al 2011). Additional physiotherapy services can be provided by health services on the weekends to increase physiotherapy contact, which may reduce length of stay and increase efficiency (Brusco et al 2007). Although providing extra physiotherapy may improve patient outcomes, little is known about how patients feel about receiving or not receiving extra physiotherapy rehabilitation services. Patient perceptions and attitudes are important because they may influence the outcomes of rehabilitation (Ohman 2005). Therefore, to provide effective rehabilitation, physiotherapists need to be aware of the elements of rehabilitation that are important to their patients (Galvin et al 2009). Previous qualitative research conducted on the experience of physiotherapy in stroke units suggests that patients would often like more physiotherapy than they receive (Galvin et al 2009, Lewinter and Mikkelsen 1995) and that an area of dissatisfaction identified by patients and their carers was the amount of physiotherapy (Wiles et al 2002).

Treatment with lambda protein phosphatase led to quantitative conversion of the 85 kDa form of SAD-A protein to the 76 kDa form (Figure 6D), indicating that SADs are phosphorylated at sites that control their activation state. We then examined a phosphoproteomic database of mouse tissues (Phosphomouse; Huttlin

et al., 2010) to identify potential sites of SAD phosphorylation. In mouse brain, SAD-A is phosphorylated on 18 sites in its C-terminal domain (CTD): 16 are proline directed, p[S/T]P, and of these, 12 are present in a striking proline-rich repeated sequence motif (PXXp[S/T]P) (Figures 6E and S6A). To determine whether these residues are phosphorylated, we expressed a SAD-A mutant in which all 18 S/T residues were mutated to nonphosphorylatable alanine (SAD-A18A). Immunoprecipitation of SAD-A followed by immunoblotting KRX-0401 with an antibody that is specific for phosphorylated Ser and Thr residues followed by Pro (p[S/T]P) Target Selective Inhibitor Library screening demonstrated that only the 85 kDa form of wild-type SAD-A was phosphorylated at S/TP motifs (Figure 6F). The SAD-A18A mutant, in contrast, migrated exclusively at 76 kDa (see lysate lanes) and was non-reactive with the p[S/T]P antibody. Thus, some or all of these 18 residues are phosphorylated in SAD-A, and this phosphorylation is a major contributor to migration

differences in SDS-PAGE. We performed two experiments to test the idea that SAD CTD phosphorylation negatively affects the ability of upstream kinases to phosphorylate the ALT site and thereby activate SAD kinase. First we immunoprecipitated SAD-AWT and SAD-A18A from HeLa cells, in which the ALT site remains unphosphorylated (see above), then added exogenous, purified LKB1 and ATP. SAD-AWT was present in both phospho-CTD (85 kDa) and dephospho-CTD (76 kDa) forms. Exogenous LKB1 phosphorylated only the 76 kDa form. SAD-A18A was present in only the 76 kDa

form, and this was significantly phosphorylated by LKB1 (Figure 6G). Second, we expressed either SAD-AWT or SAD-A18A, along with tau (a known SAD substrate, Kishi et al., 2005 and Barnes et al., 2007) in 293T cells, which have high levels of LKB1. SAD-A18A accumulated to several fold lower levels than SAD-AWT after transfection (see Discussion) but exhibited dramatically higher levels of SAD pALT phosphorylation and tau kinase activity (Figure 6H). Thus, phosphorylation Rebamipide of the SAD CTD precludes SAD kinase activation (Figure 6I). The fact that SADs are predominantly in the phospho-CTD form in neurons suggests that they are largely inactive under basal conditions. To assess mechanisms that regulate phosphorylation of the CTD, we sought CTD kinase(s). Because phosphorylation sites in the CTD are adjacent to proline residues, we treated SAD-expressing HeLa cells with inhibitors of three groups of proline-directed kinases known to play roles in neural development: MEK1/2, GSK3β and cyclin dependent kinases (CDKs) (Newbern et al.

, 2000), but its possible role in guidance of precrossing commissural axons was never investigated. However, a Sema3E gradient failed to induce turning of commissural AZD8055 supplier axons in the Dunn chamber turning assay (Figure S4A). Altogether, these results suggest that Flk1-dependent commissural axon guidance in vivo

does not occur via Sema3E and that VEGF, but not VEGF-C, is the guidance cue responsible for this effect. Floor plate-derived guidance cues such as Netrin-1 and Shh induce local changes at the growth cone in a transcriptionally independent manner (Li et al., 2004 and Yam et al., 2009). In particular, Src family kinases (SFKs) are expressed by commissural neurons and activated in their growth cones (Yam et al., 2009). Moreover, SFKs are known to participate in the guidance of axons by Netrin-1 and Shh (Li et al., 2004 and Yam et al., 2009), whereas VEGF stimulates endothelial cell

migration via SFK Ixazomib nmr activation (Eliceiri et al., 2002 and Olsson et al., 2006). Because of all these reasons, we explored whether SFKs also participated in VEGF-mediated axon guidance. Notably, VEGF stimulation of isolated commissural neurons elevated the levels of active SFKs, as measured by immunoblotting when using an antibody specifically recognizing the phosphorylated tyrosine residue Y418 in SFKs (Figure 6A). Moreover, immunostaining revealed that SFKs were activated in the growth cone (Figure 6B). Morphometric quantification revealed that VEGF, at concentrations that induced axon turning, increased the levels of phospho-SFKs in commissural neuron growth cones (Figure 6B). We next tested whether activation of SFKs is required

for VEGF-mediated axon guidance. We therefore exposed commissural neurons in the Dunn chamber to a gradient of VEGF in the presence of PP2 (a widely used SFK inhibitor) or its inactive analog (PP3). Analysis of growth cone turning revealed that neurons in the presence of PP3 turned normally in response to VEGF Levetiracetam (Figures 6C, 6D, and 6F). However, when neurons were exposed to a VEGF gradient in the presence of PP2, axons did no longer turn toward the VEGF gradient (Figures 6C, 6E, and 6F). Altogether, these results indicate that VEGF activates SFKs in commissural neurons and that SFK activity is required for VEGF-mediated commissural axon guidance. In order to reach the floor plate, commissural axons need to grow and navigate from the dorsal to the ventral spinal cord. Whereas Netrin-1 seems to account for the majority of the growth-promoting activity of the floor plate (Serafini et al., 1996), chemoattraction of precrossing commissural axons to the floor plate is controlled by both Netrin-1 and Shh (Charron et al., 2003). In the present study, we identified VEGF as an additional commissural axon chemoattractant at the floor plate. Our findings indicate that the prototypic endothelial growth factor VEGF is an axonal chemoattractant.

, 2007, Barnes and Polleux, 2009, Causeret et al., 2009, Konno et al., 2005, Sapir et al., 2008 and Solecki et al., 2004). The migration phenotypes associated with misregulation or inhibition of these genes often coincide with a multipolar or aberrant morphology in the stalled neurons. In view of these observations, in some instances it is challenging to determine whether the failure of neurons to polarize precedes the migration defects, or whether the inverse relationship holds. Notably, postmitotic granule neurons

of the cerebellum undergo axo-dendritic polarization before the onset of radial migration. In this sense, Tofacitinib chemical structure cerebellar granule neurons provide a simpler system for the study of signaling pathways specific for migration or polarity. Taking advantage of this experimental system, a recent study has uncovered that FOXO1 and the transcriptional regulator SnoN play key roles in the migration and positioning of granule neurons in the cerebellar cortex (Figure 3; Huynh et al., 2011). Alternative splicing generates two isoforms of the SnoN protein, SnoN1 and SnoN2, which differ by a 46 amino acid region present only in SnoN1 (Pearson-White and Crittenden, 1997 and Pelzer et al., 1996). PARP inhibitor SnoN1

has an essential function in limiting the extent of migration of granule neurons within the IGL and thus in the correct positioning of granule neurons within the IGL. Specific knockdown of SnoN1 in granule neurons in vivo results in abnormal accumulation of granule neurons within the deep IGL close to the white matter (Huynh et al., 2011). By contrast, SnoN2 promotes the migration of granule neurons from the EGL to the IGL. Accordingly, SnoN2 knockdown impairs migration into the IGL, leading to the accumulation of granule neurons in the EGL (Huynh et al., 2011).

Therefore, SnoN1 and SnoN2 have opposing functions in the control of granule neuron migration (Figure 3). The SnoN isoforms control migration in part by regulating the expression of the X-linked mental retardation and epilepsy gene encoding doublecortin (Dcx). Dcx promotes microtubule stability and polymerization and is thought to be critical for the dynamic coupling between the nucleus and the centrosome either during nucleokinesis (Gleeson et al., 1999, Horesh et al., 1999 and Koizumi et al., 2006). SnoN1 forms a transcriptional complex with FOXO1 that occupies the Dcx gene and thereby represses its expression in neurons (Figure 3; Huynh et al., 2011). Consistent with these findings, knockdown of the SnoN1-FOXO1 complex derepresses Dcx expression and hence stimulates excessive migration of granule neurons within the IGL in the cerebellar cortex (Huynh et al., 2011). SnoN2 antagonizes SnoN1 function by associating with SnoN1 via a coiled-coil domain interaction and inhibiting the ability of SnoN1 to repress FOXO1-dependent transcription (Figure 3; Huynh et al., 2011).

, 2006). This manipulation increased the OSD and MT (Figure 2Aiii; Table 1) but again failed to induce an increase in accuracy (Figure 2Aii; Table 1). Therefore, we next tested the effects of increasing the incentive to obtain correct responses by eliminating water outside the task, increasing task difficulty and decreasing the number of available trials (see Experimental Procedures for details). Although this manipulation produced a drop in body weight of test subjects compared to controls (Figure 2Bi) demonstrating its effectiveness, there was no difference in accuracy,

OSD or MT between test and control MLN0128 molecular weight groups (Figures 2Bii and 2Biii; Table 1). To directly assess the impact of differential reward AT13387 ic50 expectation on measures of response time, we trained another set of rats on a one-direction-rewarded (1DR) version of the two-alternative choice task. In this task

version, only responses to one choice direction were rewarded (when correct) and this rewarded direction changed across blocks within a session. As expected, animals were biased to choose the rewarded side (Figure 2Ci) and performance increased for the rewarded side for the difficult odor mixtures (Figure 2Cii). We found that OSD for nonrewarded choices was slower than for the rewarded ones (Figure 2Ciii). Moreover the effect of stimulus difficulty on OSD was diminished for the nonrewarded choices (Figure 2Ciii), those choices whose difficulty no longer predicted the likelihood of reward availability. These results suggest that the effect of difficulty on OSD arises not only from varying perceptual uncertainty but also reflects the effect of difficulty on reward Adenylyl cyclase expectation and hence response speed. Having seen that response times are sensitive to reward and punishment but that changes in OSD did not produce significant changes in accuracy, we next sought to test the possible effect of larger changes in stimulus sampling time by manipulating the OSD more directly. To do so, following

a previous study (Rinberg et al., 2006), we introduced an auditory go signal to cue the timing of the response while the odor stimulus continued to cue the correct choice direction (Figure 3A). Responses initiated prior to the go signal were not rewarded regardless of choice. We first trained subjects to wait for the go signal (see Experimental Procedures). After training, we used fixed go-signal delays of 0, 0.2, 0.4, and 0.8 s, each repeated for 3–6 sessions before switching (Figure 3B). Within each session odor mixtures of the same difficulties as the RT task were randomly interleaved from trial-to-trial. Subjects obeyed the go signal, resulting in much longer OSDs than those seen in the original RT task (Figure 3C; Table 1). However, despite the substantial increase in odor sampling durations, we observed no change in accuracy (Figure 3D; Table 1; Figure S3).