Development of a mobile, high-throughput, and low-cost image-based plant growth phenotyping system.

Nondestructive plant phenotyping forms a key technique for unraveling molecular processes underlying plant development and response to the environment. While the emergence of high-throughput phenotyping facilities can further our understanding of plant development and stress responses, their high costs greatly hinder scientific progress. To democratize high-throughput plant phenotyping, we developed sets of low-cost image- and weight-based devices to monitor plant shoot growth and evapotranspiration. We paired these devices to a suite of computational pipelines for integrated and straightforward data analysis. The developed tools were validated for their suitability for large genetic screens by evaluating a cowpea (Vigna unguiculata) diversity panel for responses to drought stress. The observed natural variation was used as an input for a genome-wide association study, from which we identified nine genetic loci that might contribute to cowpea drought resilience during early vegetative development. The homologs of the candidate genes were identified in Arabidopsis (Arabidopsis thaliana) and subsequently evaluated for their involvement in drought stress by using available T-DNA insertion mutant lines. These results demonstrate the varied applicability of this low-cost phenotyping system. In the future, we foresee these setups facilitating the identification of genetic components of growth, plant architecture, and stress tolerance across a wide variety of plant species.

Improving Phosphoproteomics Profiling Using Data-Independent Mass Spectrometry.

Mass spectrometry-based profiling of the phosphoproteome is a powerful method of identifying phosphorylation events at a systems level. Most phosphoproteomics studies have used data-dependent acquisition (DDA) mass spectrometry as their method of choice. In this Perspective, we review some recent studies benchmarking DDA and DIA methods for phosphoproteomics and discuss data analysis options for DIA phosphoproteomics. In order to evaluate the impact of data-dependent and data-independent acquisition (DIA) on identification and quantification, we analyze a previously published phosphopeptide-enriched data set consisting of 10 replicates acquired by DDA and DIA each. We find that though more unique identifications are made in DDA data, phosphopeptides are identified more consistently across replicates in DIA. We further discuss the challenges of identifying chromatographically coeluting phosphopeptide isomers and investigate the impact on reproducibility of identifying high-confidence site-localized phosphopeptides in replicates.

Echocardiographic linear fractional shortening for quantification of right ventricular systolic function-A cardiac magnetic resonance validation study.

BACKGROUND: Echocardiography (echo)-based linear fractional shortening (FS) is widely used to assess left ventricular dysfunction (LVdys ), but has not been systematically tested for right ventricular dysfunction (RVdys ). METHODS: The population comprised LVdys patients with and without RVdys (EF<50%) on cardiac MRI (CMR): Echo included standard RV indices (fractional area change [FAC], TAPSE, S', and FS in parasternal long-axis (RV outflow tract [RVOT ]) and apical four-chamber views (width [RVWD ], length [RVLG ]). RESULTS: A total of 168 patients underwent echo and CMR (3±3 days); FAC (46±9 vs 28±11), TAPSE (1.9±0.4 vs 1.5±0.3), and S' (11.4±2.3 vs 10.0±2.6, all P≤.001) were lower among RVdys patients, as were FS indices (RVOT 32±8 vs 17±10 | RVWD 40±11 vs 22±12 | RVLG 16±5 vs 9±4%; all P<.001). FS indices yielded similar magnitude of correlation with CMR RVEF (r=.73-.56) as did FAC (r=.70), which was slightly higher than TAPSE (r=.47) and S' (r=.31; all P<.001). FS indices decreased stepwise vs CMR RVEF tertiles, as did FAC (all P<.001). In multivariate analysis, FS in RVOT (regression coefficient .51 [CI 0.37-0.65]), RVWD (0.30 [0.19-0.41]), and RVLG (0.45 [0.20-0.71]; all P≤.001) was independently associated with CMR RVEF. FS indices yielded good overall diagnostic performance (AUC: RVOT 0.89 [CI 0.82-0.97] | RVWD 0.87 [0.78-0.96] | RVLG 0.80 [0.70-0.90]; all P<.001) for CMR-defined RVdy (RVEF<50%). CONCLUSIONS: RV linear FS provides RV functional indices that parallel CMR RVEF. Parasternal long-axis RVOT width, four-chamber RV width, and length are independently associated with RVEF, supporting use of multiple FS indices for RV functional assessment.

Echocardiography-quantified myocardial strain-a marker of global and regional infarct size that stratifies likelihood of left ventricular thrombus.

BACKGROUND: Myocardial strain provides a novel means of quantifying subtle alterations in contractile function; incremental utility post-MI is unknown. OBJECTIVES: To test longitudinal-quantified by postprocessing routine echo-for assessment of MI size measured by cardiac magnetic resonance (CMR) and conventional methods, and assess regional and global strain (GLS) as markers of LV thrombus. METHODS: The population comprised of patients with anterior ST-segment MI who underwent echo and CMR prospectively. Preexisting echoes were retrieved, re-analyzed for strain, and compared to conventional MI markers as well as CMR-evidenced MI, function, and thrombus. RESULTS: Seventy-four patients underwent echo and CMR 4 ± 1 weeks post-MI; 72% had abnormal GLS. CMR-quantified MI size was 2.5-fold larger and EF lower among patients with abnormal GLS, paralleling 2.6-3.1 fold differences in Q-wave size and CPK (all P ≤ .002). GLS correlated with CMR-quantified MI (r = .66), CPK (r = .52) and Q-wave area (r = .44; all P ≤ .001): Regional strain was lower in the base, mid, and apical LV among patients with CMR-defined transmural MI in each territory (P < .05) and correlated with cine-CMR regional EF (r = .53-.71; P < .001) and echo wall motion (r = .45-.71; P < .001). GLS and apical strain were ~2-fold lower among patients with LV thrombus (P ≤ .002): Apical strain yielded higher diagnostic performance for thrombus (AUC: 0.83 [0.72-0.93], P = .001) than wall motion (0.73 [0.58-0.88], P = .02), as did global strain (0.78 [0.65-0.90], P = .005) compared to LVEF (0.58 [0.45-0.72], P = .41). CONCLUSIONS: Echo-quantified longitudinal strain provides a marker of MI size and improves stratification for post-MI LV thrombus beyond conventional indices.

An accurate and rapidly calibrating speech neuroprosthesis.

Brain-computer interfaces can enable rapid, intuitive communication for people with paralysis by transforming the cortical activity associated with attempted speech into text on a computer screen. Despite recent advances, communication with brain-computer interfaces has been restricted by extensive training data requirements and inaccurate word output. A man in his 40's with ALS with tetraparesis and severe dysarthria (ALSFRS-R = 23) was enrolled into the BrainGate2 clinical trial. He underwent surgical implantation of four microelectrode arrays into his left precentral gyrus, which recorded neural activity from 256 intracortical electrodes. We report a speech neuroprosthesis that decoded his neural activity as he attempted to speak in both prompted and unstructured conversational settings. Decoded words were displayed on a screen, then vocalized using text-to-speech software designed to sound like his pre-ALS voice. On the first day of system use, following 30 minutes of attempted speech training data, the neuroprosthesis achieved 99.6% accuracy with a 50-word vocabulary. On the second day, the size of the possible output vocabulary increased to 125,000 words, and, after 1.4 additional hours of training data, the neuroprosthesis achieved 90.2% accuracy. With further training data, the neuroprosthesis sustained 97.5% accuracy beyond eight months after surgical implantation. The participant has used the neuroprosthesis to communicate in self-paced conversations for over 248 hours. In an individual with ALS and severe dysarthria, an intracortical speech neuroprosthesis reached a level of performance suitable to restore naturalistic communication after a brief training period.

Increased adipose catecholamine levels and protection from obesity with loss of Allograft Inflammatory Factor-1.

Recent studies implicate macrophages in regulation of thermogenic, sympathetic neuron-mediated norepinephrine (NE) signaling in adipose tissues, but understanding of such non-classical macrophage activities is incomplete. Here we show that male mice lacking the allograft inflammatory factor-1 (AIF1) protein resist high fat diet (HFD)-induced obesity and hyperglycemia. We link this phenotype to higher adipose NE levels that stem from decreased monoamine oxidase A (MAOA) expression and NE clearance by AIF1-deficient macrophages, and find through reciprocal bone marrow transplantation that donor Aif1-/- vs WT genotype confers the obesity phenotype in mice. Interestingly, human sequence variants near the AIF1 locus associate with obesity and diabetes; in adipose samples from participants with obesity, we observe direct correlation of AIF1 and MAOA transcript levels. These findings identify AIF1 as a regulator of MAOA expression in macrophages and catecholamine activity in adipose tissues - limiting energy expenditure and promoting energy storage - and suggest how it might contribute to human obesity.

Culling of activated CD4 T cells during typhoid is driven by Salmonella virulence genes.

Pathogen-specific CD4 T cells are activated within a few hours of oral Salmonella infection and are essential for protective immunity. However, CD4 T cells do not participate in bacterial clearance until several weeks after infection, suggesting that Salmonella can inhibit or evade CD4 T cells that are activated at early time points. Here, we describe the progressive culling of initially activated CD4 T cells in Salmonella-infected mice. Loss of activated CD4 T cells was independent of early instructional programming, T cell precursor frequency, and Ag availability. In contrast, apoptosis of Ag-specific CD4 T cells was actively induced by live bacteria in a process that required Salmonella pathogenicity island-2 and correlated with increased expression of PD-L1. These data demonstrate efficient culling of initially activated Ag-specific CD4 cells by a microbial pathogen and document a novel strategy for bacterial immune evasion.

Analysis of Relative Scent Intensity, Volatile Compounds and Gene Expression in Freesia "Shiny Gold".

Scent is one of the most important economic traits in Freesia hybrida. "Shiny Gold", a popular cultivar in South Korea, is widely cultivated for its scent. The relative scent intensity of "Shiny Gold" was approximately 16% higher in full-bloomed flower when compared to the yellow bud stage, while tissue-specifically, tepals showed higher intensity in electronic-nose (e-nose) analysis. E-nose analysis also showed that the scent intensity of "Shiny Gold" was higher and lower than "10C3-424" and "10C3-894", respectively, and was similar to "Yvonne". These results correlated to those of the olfactory tests. In total, 19 volatile compounds, including linalool, ß-ocimene, D-limonene, trans-ß-ionone were detected in gas chromatography-mass spectrometry analysis. Among these, linalool was the major volatile compound, accounting for 38.7% in "Shiny Gold". Linalool synthase and TPS gene expression corresponded to the scent intensity of the four cultivars, with the lowest expression in the "10C3-424". TPS 2, TPS 3, TPS 5, TPS 6 and TPS 8 were highly expressed in both bud and flower in "Shiny Gold", while the expression of TPS 4 was lower, relative to other TPS genes in both the flowering stages. These results may aid in enhancing scent composition in Freesia cultivars using marker-assisted selection.

Expression of Toll/IL-1R domain-containing adaptor protein (TIRAP) is detrimental to primary clearance of Salmonella and is not required for the generation of protective immunity.

Salmonella infection triggers activation of innate immune cells through the interaction of bacterial products with Toll-like receptors (TLRs). Toll/IL-1R domain-containing adaptor protein (TIRAP) is an adaptor protein involved in downstream signaling through TLRs 1, 2, 4, and 6. We examined the role of TIRAP during infection with attenuated Salmonella. Surprisingly, TIRAP-deficient mice were fully capable of resolving primary infection with Salmonella and actually exhibited accelerated clearance of bacteria at a late stage of the infection. Consistent with enhanced bacterial clearance, TIRAP-deficient mice resolved bacterial-associated splenic inflammation more rapidly than wild-type (Wt) mice and splenocytes from Salmonella-infected TIRAP-deficient mice produced more IFN-gamma upon in vitro re-stimulation. Upon secondary challenge, TIRAP-deficient and Wt mice displayed a similar level of protective immunity against virulent Salmonella. Together these data indicate that TIRAP-mediated signaling is completely dispensable for clearance of Salmonella infection, and actually has a mild deleterious effect upon the resolution of primary infection.

Pivotal advance: exposure to LPS suppresses CD4+ T cell cytokine production in Salmonella-infected mice and exacerbates murine typhoid.

A number of studies have documented suppression of lymphocyte activation in mice infected with Salmonella. Here, we describe incomplete activation of CD4+ T cells following intravenous injection of specific peptide and LPS into Salmonella-infected mice. Although antigen-specific CD4+ T cells were activated by peptide/LPS to increase surface CD69 expression, they did not produce IL-2 or TNF-alpha. Suppression of cytokine production did not require prolonged exposure of the T cells to the Salmonella-infected environment, was not antigen specific, but was dependent upon the presence of LPS during stimulation. These data suggest that Salmonella-infected mice are exquisitely sensitive to the generation of a suppressive environment following innate immune stimulation with LPS. In agreement with this interpretation, repeated low-dose administration of LPS caused uncontrolled replication of attenuated Salmonella in vivo.

Sinonasal Teratocarcinosarcoma- A Rare Tumour not so Rarely Misdiagnosed.

Sinonasal teratocarcinosarcoma (STCS) is a rare, morphologically heterogeneous and highly aggressive tumour of ambiguous origin. It is characterized by the presence of benign and malignant epithelial, mesenchymal and neuroectodermal components. Because of their rarity and heterogeneity, these lesions are often misdiagnosed, leading to management difficulties. Adequate sampling with a high index of suspicion is needed to diagnose this rare tumour. We reported here a 48-year old man with right nasoethmoidal mass eroding the cribriform plate with intracranial extension. An initial incisional biopsy was performed and a diagnosis of craniopharyngioma was made. Subtotal endoscopic excision of the mass revealed features of STCS. Immunohistochemistry confirmed the same. The patient was subsequently treated with radiotherapy. The histogenesis, histopathological features, immunohistochemistry findings, clinical features and treatment were discussed here. Till date, there are less than 100 cases reported in English literature.

Visualizing the immune response to pathogens.

Advances in immune visualization have enabled the physical tracking of immune responses in vivo. The adaptation of such technology to models of infectious disease holds the promise of a more detailed analysis of host-pathogen interactions in a natural setting. However, the visualization of pathogen-specific immune responses in vivo confronts challenges that are inherent to the study of infectious disease systems. Recent attempts to track pathogen-specific immune responses in vivo validate the usefulness and underline the complexity of this experimental strategy.

Multiplanar strain quantification for assessment of right ventricular dysfunction and non-ischemic fibrosis among patients with ischemic mitral regurgitation.

BACKGROUND: Ischemic mitral regurgitation (iMR) predisposes to right ventricular (RV) pressure and volume overload, providing a nidus for RV dysfunction (RVDYS) and non-ischemic fibrosis (NIF). Echocardiography (echo) is widely used to assess iMR, but performance of different indices as markers of RVDYS and NIF is unknown. METHODS: iMR patients prospectively underwent echo and cardiac magnetic resonance (CMR) within 72 hours. Echo quantified iMR, assessed conventional RV indices (TAPSE, RV-S', fractional area change [FAC]), and strain via speckle tracking in apical 4-chamber (global longitudinal strain [RV-GLS]) and parasternal long axis orientation (transverse strain). CMR volumetrically quantified RVEF, and assessed ischemic pattern myocardial infarction (MI) and septal NIF. RESULTS: 73 iMR patients were studied; 36% had RVDYS (EF<50%) on CMR among whom LVEF was lower, PA systolic pressure higher, and MI size larger (all p<0.05). CMR RVEF was paralleled by echo results; correlations were highest for RV-GLS (r = 0.73) and lowest for RV-S' (r = 0.43; all p<0.001). RVDYS patients more often had CMR-evidenced NIF (54% vs. 7%; p<0.001). Whereas all RV indices were lower among NIF-affected patients (all p≤0.006), percent change was largest for transverse strain (48.3%). CMR RVEF was independently associated with RV-GLS (partial r = 0.57, p<0.001) and transverse strain (r = 0.38, p = 0.002) (R = 0.78, p<0.001). Overall diagnostic performance of RV-GLS and transverse strain were similar (AUC = 0.93[0.87-0.99]|0.91[0.84-0.99], both p<0.001), and yielded near equivalent sensitivity and specificity (85%|83% and 80%|79% respectively). CONCLUSION: Compared to conventional echo indices, RV strain parameters yield stronger correlation with CMR-defined RVEF and potentially constitute better markers of CMR-evidenced NIF in iMR.

Activation of Salmonella-specific immune responses in the intestinal mucosa.

The mammalian immune response to Salmonella has long been a subject of scientific study. Indeed, many of the general aspects of bacterial pathogenesis and host immune defense have been well described. However, a lack of clarity remains concerning important aspects of the host immune response to Salmonella, particularly with regard to the induction of an immune response in the intestinal mucosa. A major limitation has been the general lack of knowledge about specific antigenic targets that are recognized by both the innate and adaptive immune response in the intestine. Progress towards the identification of these targets is critical for the development of a detailed model of immunity to Salmonella and will lead to a better understanding of mucosal immune responses to other intracellular pathogens.

Echocardiographic Linear Dimensions for Assessment of Right Ventricular Chamber Volume as Demonstrated by Cardiac Magnetic Resonance.

BACKGROUND: Echocardiography-derived linear dimensions offer straightforward indices of right ventricular (RV) structure but have not been systematically compared with RV volumes on cardiac magnetic resonance (CMR). METHODS: Echocardiography and CMR were interpreted among patients with coronary artery disease imaged via prospective (90%) and retrospective (10%) registries. For echocardiography, American Society of Echocardiography-recommended RV dimensions were measured in apical four-chamber (basal RV width, mid RV width, and RV length), parasternal long-axis (proximal RV outflow tract [RVOT]), and short-axis (distal RVOT) views. For CMR, RV end-diastolic volume and RV end-systolic volume were quantified using border planimetry. RESULTS: Two hundred seventy-two patients underwent echocardiography and CMR within a narrow interval (0.4 ± 1.0 days); complete acquisition of all American Society of Echocardiography-recommended dimensions was feasible in 98%. All echocardiographic dimensions differed between patients with and those without RV dilation on CMR (P < .05). Basal RV width (r = 0.70), proximal RVOT width (r = 0.68), and RV length (r = 0.61) yielded the highest correlations with RV end-diastolic volume on CMR; end-systolic dimensions yielded similar correlations (r = 0.68, r = 0.66, and r = 0.65, respectively). In multivariate regression, basal RV width (regression coefficient = 1.96 per mm; 95% CI, 1.22-2.70; P < .001), RV length (regression coefficient = 0.97; 95% CI, 0.56-1.37; P < .001), and proximal RVOT width (regression coefficient = 2.62; 95% CI, 1.79-3.44; P < .001) were independently associated with CMR RV end-diastolic volume (r = 0.80). RV end-systolic volume was similarly associated with echocardiographic dimensions (basal RV width: 1.59 per mm [95% CI, 1.06-2.13], P < .001; RV length: 1.00 [95% CI, 0.66-1.34], P < .001; proximal RVOT width: 1.80 [95% CI, 1.22-2.39], P < .001) (r = 0.79). CONCLUSIONS: RV linear dimensions provide readily obtainable markers of RV chamber size. Proximal RVOT and basal width are independently associated with CMR volumes, supporting the use of multiple linear dimensions when assessing RV size on echocardiography.

Right Ventricular Dysfunction Impairs Effort Tolerance Independent of Left Ventricular Function Among Patients Undergoing Exercise Stress Myocardial Perfusion Imaging.

BACKGROUND: Right ventricular (RV) and left ventricular (LV) function are closely linked due to a variety of factors, including common coronary blood supply. Altered LV perfusion holds the potential to affect the RV, but links between LV ischemia and RV performance, and independent impact of RV dysfunction on effort tolerance, are unknown. METHODS AND RESULTS: The population comprised 2051 patients who underwent exercise stress myocardial perfusion imaging and echo (5.5±7.9 days), among whom 6% had echo-evidenced RV dysfunction. Global summed stress scores were ≈3-fold higher among patients with RV dysfunction, attributable to increments in inducible and fixed LV perfusion defects (all P≤0.001). Regional inferior and lateral wall ischemia was greater among patients with RV dysfunction (both P<0.01), without difference in corresponding anterior defects (P=0.13). In multivariable analysis, inducible inferior and lateral wall perfusion defects increased the likelihood of RV dysfunction (both P<0.05) independent of LV function, fixed perfusion defects, and pulmonary artery pressure. Patients with RV dysfunction demonstrated lesser effort tolerance whether measured by exercise duration (6.7±2.8 versus 7.9±2.9 minutes; P<0.001) or peak treadmill stage (2.6±0.9 versus 3.1±1.0; P<0.001), paralleling results among patients with LV dysfunction (7.0±2.9 versus 8.0±2.9; P<0.001|2.7±1.0 versus 3.1±1.0; P<0.001 respectively). Exercise time decreased stepwise in relation to both RV and LV dysfunction (P<0.001) and was associated with each parameter independent of age or medication regimen. CONCLUSIONS: Among patients with known or suspected coronary artery disease, regional LV ischemia involving the inferior and lateral walls confers increased likelihood of RV dysfunction. RV dysfunction impairs exercise tolerance independent of LV dysfunction.

Performance of a new meter designed for assisted monitoring of blood glucose and point-of-care testing.

BACKGROUND: Blood glucose (BG) meters used for assisted monitoring of blood glucose (AMBG) require different attributes compared with meters designed for home use. These include safety considerations (i.e., minimized risk of blood-borne pathogen transmission), capability for testing multiple blood sample types, and enhanced performance specifications. The OneTouch® Verio™Pro+ BG meter is designed to incorporate all of these attributes. METHODS: Meter accuracy was assessed in clinical studies with arterial, venous, and capillary blood samples with a hematocrit range of 22.9-59.8%. The effect of interferents, including anticoagulants, on accuracy was evaluated. The meter disinfection protocol was validated, and instructions for use and user acceptance of the system were assessed. RESULTS: A total of 97% (549/566) of BG measures from all blood sample types and 95.5% (191/200) of arterial blood samples were within ±12 mg/dl or 12.5% of reference measurements. The system was unaffected by 4 anticoagulants and 57 of 59 endogenous and exogenous compounds; it was affected by 2 compounds: pralidoxime iodide and xylose. Bleach wipes were sufficient to disinfect the meter. Users felt that the meter's quality control (QC) prompts would help them to comply with regulatory requirements. CONCLUSION: The meter provided accurate measurements of different blood samples over a wide hematocrit range and was not affected by 57 physiologic and therapeutic compounds. The QC prompts and specific infection-mitigating design further aid to make this meter system practical for AMBG in care facilities.

Evaluation of a blood glucose monitoring system with automatic high- and low-pattern recognition software in insulin-using patients: pattern detection and patient-reported insights.

BACKGROUND: This study aimed to evaluate the performance of a glucose pattern recognition tool incorporated in a blood glucose monitoring system (BGMS) and its association with clinical measures, and to assess user perception and understanding of the pattern messages they receive. METHODS: Participants had type 1 or type 2 diabetes mellitus and were self-adjusting insulin doses for ≥1 year. During a 4-week home testing period, participants performed ≥6 daily self-tests, adjusted their insulin regimen based on BGMS results, and recorded pattern messages in the logbook. Participants reflected on usability of the pattern tool in a questionnaire. RESULTS: Study participants (n = 101) received a mean ± standard deviation of 4.5 ± 1.9 pattern messages per week (3.6 ± 1.8 high glucose patterns and 0.9 ± 1.3 low glucose patterns). Most received ≥1 high (96.5%) and/or ≥1 low (46.0%) pattern message per week. The average number of high- and low-pattern messages per week was associated with higher and lower, respectively, baseline hemoglobin A1c (p < .01) and fasting plasma glucose (p < .05). Participants found high- and low-pattern messages clear and easy to understand (84.2% and 83.2%, respectively) and considered the frequency of low (82.0%) and high (63.4%) pattern messages about right. Overall, 71.3% of participants indicated they preferred to use a meter with pattern messages. CONCLUSIONS: The on-device Pattern tool identified meaningful blood glucose patterns, highlighting potential opportunities for improving glycemic control in patients who self-adjust their insulin.

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18.

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.