A proprioceptive feedback circuit drives Caenorhabditis elegans locomotor adaptation through dopamine signaling.

An animal adapts its motor behavior to navigate the external environment. This adaptation depends on proprioception, which provides feedback on an animal's body postures. How proprioception mechanisms interact with motor circuits and contribute to locomotor adaptation remains unclear. Here, we describe and characterize proprioception-mediated homeostatic control of undulatory movement in the roundworm Caenorhabditis elegans. We found that the worm responds to optogenetically or mechanically induced decreases in midbody bending amplitude by increasing its anterior amplitude. Conversely, it responds to increased midbody amplitude by decreasing the anterior amplitude. Using genetics, microfluidic and optogenetic perturbation response analyses, and optical neurophysiology, we elucidated the neural circuit underlying this compensatory postural response. The dopaminergic PDE neurons proprioceptively sense midbody bending and signal to AVK interneurons via the D2-like dopamine receptor DOP-3. The FMRFamide-like neuropeptide FLP-1, released by AVK, regulates SMB head motor neurons to modulate anterior bending. We propose that this homeostatic behavioral control optimizes locomotor efficiency. Our findings demonstrate a mechanism in which proprioception works with dopamine and neuropeptide signaling to mediate motor control, a motif that may be conserved in other animals.

C─H Activation Enables the Construction of New Bis-Polyaryl Phenylpyridine Ruthenium Complexes: Conjugation and Rigidity Synergistic Effect for Advanced Electrochemiluminescence.

The access to bench-stable organometallic compounds unfolds new chemical space for medicinal and material sciences. In particular, stable organoruthenium compounds with constitutional and stereoisomeric forms for subtle regulation of electrochemiluminescence are intriguing and challenging. Here, coordination of polycyclic aromatic hydrocarbons on (2-phenylpyridine)2(CO)2Ru complex allows access to bis-polyaryl phenylpyridine (BPP) Ruthenium complex through C─H activation strategy and coupling reactions for installation of the functionalities with steric and electronic purposes. The photoluminescence and electrochemiluminescence of BPP Ru complexes are affected by the actual polycyclic aromatic hydrocarbons inherent properties. The anthracene derivatized BPP Ru complex (BPP-Ant) shows the best ECL performance and reveals an enormous ECL quantum efficiency of 1.6-fold higher than the golden standard Ru(bpy)3 2+. The unprecedentedly high efficiency is due to the best compromise between the structural conjugation and molecular rigidity from BPP-Ant providing a providential energy gap that facilitated the feasibility of electron transfer and favored the radiative energy release by experimentally and DFT calculations. Moreover, PL and spooling ECL spectroscopies are used to track and link multiple emission peaks of BPP-Ant at 445, 645, and 845 nm to different emissive species. These discoveries will add a new member to the efficient ECL ruthenium complex family and bring more potentials.

A new modular framework for high-level application development at HEPS.

As a representative of the fourth-generation light sources, the High Energy Photon Source (HEPS) in Beijing, China, utilizes a multi-bend achromat lattice to obtain an approximately 100 times emittance reduction compared with third-generation light sources. New technologies bring new challenges to operate the storage ring. In order to meet the beam commissioning requirements of HEPS, a new framework for the development of high-level applications (HLAs) has been created. The key part of the new framework is a dual-layer physical module to facilitate the seamless fusion of physical simulation models with the real machine, allowing for fast switching between different simulation models to accommodate the various simulation scenarios. As a framework designed for development of physical applications, all variables are based on physical quantities. This allows physicists to analytically assess measurement parameters and optimize machine parameters in a more intuitive manner. To enhance both extensibility and adaptability, a modular design strategy is utilized, partitioning the entire framework into discrete modules in alignment with the requirements of HLA development. This strategy not only facilitates the independent development of each module but also minimizes inter-module coupling, thereby simplifying the maintenance and expansion of the entire framework. To simplify the development complexity, the design of the new framework is implemented using Python and is called Python-based Accelerator Physics Application Set (Pyapas). Taking advantage of Python's flexibility and robust library support, we are able to develop and iterate quickly, while also allowing for seamless integration with other scientific computing applications. HLAs for both the HEPS linac and booster have been successfully developed. During the beam commissioning process at the linac, Pyapas's ease of use and reliability have significantly reduced the time required for the beam commissioning operators. As a development framework for HLA designed for the new-generation light sources, Pyapas has the versatility to be employed with HEPS, as well as with other comparable light sources, due to its adaptability.

Serum amyloid A 1 induces suppressive neutrophils through the Toll-like receptor 2-mediated signaling pathway to promote progression of breast cancer.

Immune inflammation plays a key role in breast cancer development, progression, and therapeutic efficacy. Neutrophils are crucial for the regulation of the suppressive tumor microenvironment and are associated with poor clinical survival. However, the mechanisms underlying the activation of suppressive neutrophils in breast cancer are poorly understood. Here, we report that breast cancer cells secrete abundant serum amyloid A 1 (SAA1), which is associated with the accumulation of suppressive neutrophils. High expression of SAA1 in breast cancer induces neutrophil immunosuppressive cytokine production through the activation of Toll-like receptor 2 (TLR2)-mediated signaling pathways. These include the TLR2/myeloid differentiation primary response 88 (MYD88)-mediated PI3K/nuclear factor-κB signaling pathway and p38 MAPK-associated apoptosis resistance pathway, which eventually promote the progression of breast cancer. Our study shows a mechanistic link between breast cancer cell secretion of SAA1 and suppressive neutrophils that potentiate tumor progression. These findings provide potential therapeutic targets for breast cancer.

HMGB1 is a key factor for tamoxifen resistance and has the potential to predict the efficacy of CDK4/6 inhibitors in breast cancer.

Breast cancer is the leading cause of cancer death in women. Hormone-receptor-positive breast cancer (HR + BC) is the most common pathological type of breast cancer, of which the main treatment method is endocrine therapy. Unfortunately, primary or acquired drug resistance greatly limits its efficacy. In recent years, the newly launched CDK4/6 inhibitors could effectively reverse endocrine resistance in breast cancer. However, considering their expensive price and side effects, it is particularly important to find out effective biomarkers and screen sensitive patients. Here, we found through bioinformatics analysis that high mobility group box 1 (HMGB1) expression increased in endocrine-resistant HR + BC. In clinical specimens, the higher expression of HMGB1 was associated with shorter progression-free survival (PFS) for HR + BC patients with endocrine therapy after surgery. For endocrine-resistant breast cancer, compared with HMGB1-negative patients, HMGB1-positive patients who received CDK4/6 inhibitors treatment benefited more in PFS. Moreover, we demonstrated that HMGB1 promoted tamoxifen resistance by combining with the Toll-like receptor 4 (TLR4) and activating nuclear factor kappa B (NF-κB) pathway. CDK4/6 inhibitors could downregulate the expression of HMGB1 and suppress the TLR4-NF-κB pathway, and in turn reverse tamoxifen resistance. These results illuminated the critical role of HMGB1 in the process of tamoxifen resistance, explained the mechanism of CDK4/6 inhibitors reversing tamoxifen resistance, and suggested the feasibility of HMGB1 as a potential biomarker for screening sensitive patients receiving CDK4/6 inhibitors.

Tumor-associated macrophages increase COX-2 expression promoting endocrine resistance in breast cancer via the PI3K/Akt/mTOR pathway.

Breast cancer is the most common malignancy in females. The emergence of endocrine resistance is frustrating for estrogen receptor (ER)-positive breast cancer patients even the efficacy of endocrine therapy is acceptable. Our previous study has shown that tumor-associated macrophages (TAMs) are associated with endocrine resistance, yet the mechanism remains unclear. This article is dedicated to discuss the role of TAMs in the endocrine resistance of breast cancer. It was found that tamoxifen-resistant MCF-7 cells induced more macrophages polarized into TAMs. Conversely, TAMs increased the expression of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2), which promoted tamoxifen resistance through the activation of the PI3K/Akt/mTOR signaling pathway in MCF-7 cells. Furthermore, clinical analysis supported that five-year progression-free survival (PFS) of breast cancer patients with abundant COX-2 expression in TAMs was shorter (p<0.05). Therefore, these results show a positive feedback loop between TAMs and breast cancer cells, suggesting that TAMs and COX-2 may be new therapeutic targets for breast cancer patients suffering from endocrine resistance.

Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer.

Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, the development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor-associated macrophages (TAM) within the TME play a crucial role in endocrine resistance. Herein, we aimed to elucidate the relationship between TAM and the endocrine-resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen-sensitive (MCF7-S) or -resistant (MCF7-R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAM, and then treated with tamoxifen. CC-chemokine ligand 2 (CCL2) immunohistochemistry was carried out on pathological sections from 100 patients with invasive estrogen receptor-positive breast cancer. We found that macrophages cultured in the CM of MCF7-S and MCF7-R cells were induced into TAM, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAM secreted CCL2, which increased endocrine resistance in breast cancer cells through activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with infiltration of CD163+macrophages (r = 0.548, P < .001), and patients with high CCL2 expression presented shorter progression-free survival than those with low CCL2 expression (P < .05). We conclude that CCL2 secreted by TAM activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAM may be novel therapeutic targets for patients with endocrine-resistant breast cancer.

LDOC1 is differentially expressed in thyroid cancer and display tumor-suppressive function in papillary thyroid carcinoma.

The leucine zipper downregulated in cancer 1 (LDOC1) has been proposed as a regulator of transcription and cell signaling. We have previously demonstrated that LDOC1 is differentially expressed in papillary thyroid carcinoma (PTC), this study was designed to characterize LDOC1 expression in thyroid follicle originated cancer tissues and to specifically evaluate its function in thyroid carcinogenesis. LDOC1 expression was performed in human normal thyroid and thyroid cancer. LDOC1 function was characterized, in two PTC cell lines (TPC1 and BCPAP), through the analysis of in vitro cell proliferation, apoptosis, migration, and invasion along with in vivo tumor xenograft growth. Transduced BCPAP cells were stimulated with tumor necrosis factor α, and the levels of nuclear P65, Bax, Bcl-2, c-Myc, and XIAP were assessed. A luciferase reporter assay was used to measure nuclear factor-κB (NF-κB) activity, and the functional connection between LDOC1 effect and NF-κB activity was determined using a specific NF-κB inhibitor. Our results revealed that LDOC1 was translocated from the nucleus to the cytoplasm in human thyroid cancer, and was significantly downregulated in PTC compared with normal thyroid. LDOC1 overexpression in TPC1 resulted in a significant suppression of the malignant phenotype, whereas LDOC1 ablation in BCPAP promoted this phenotype. Additional studies demonstrated that LDOC1 ablation facilitated nuclear P65 expression and NF-κB activity. NF-κB inhibition reversed the effects of LDOC1 ablation on proliferation, apoptosis, migration, and invasion. Our findings confirmed that LDOC1 is a novel therapeutic target in PTC and provides new insight into the role of LDOC1 in PTC progression, through NF-κΒ signaling suppression.

Pan-neuronal imaging in roaming Caenorhabditis elegans.

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.

Ratio of Immune Response to Tumor Burden Predicts Survival Via Regulating Functions of Lymphocytes and Monocytes in Diffuse Large B-Cell Lymphoma.

BACKGROUND/AIMS: Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease, and is the most common type of lymphoma in adults. Although significant progress in treatment has been made using chemotherapy combinations, there exist a large amount of relapse or refractory cases. Thus, effective clinical biomarkers for DLBCL are urgently needed. Our study aims to explore the predictive significance of using the immune response to tumor burden ratio [defined as the lymphocyte to monocyte ratio (LMR)/lactate dehydrogenase (LDH) levels] in 184 DLBCL patients and the potential mechanism underlying the use of the LMR to tumor burden ratio in predicting patient survival. METHODS: The correlation between serum LDH levels and tumor levels assessed by PET-CT was determined using Spearman's correlation analysis. Clinical data from 184 DLBCL patients was assessed using receiver operating characteristic curve analysis and survival analysis. The potential correlation between tumor burden and lymphocytes or monocytes was analyzed by immunohistochemical staining, flow cytometry, and ELISA analysis of patient samples. In addition, we performed in vitro studies to further determine the effects of tumor burden on the anti-tumor activity of T lymphocytes. RESULTS: We observed that serum LDH was an excellent surrogate marker of tumor burden in DLBCL patients, and that the ratio of LMR to LDH was an independent prognostic biomarker capable of predicting survival in DLBCL patients. Further analysis showed that a high tumor burden was correlated with decreased Ki67 expression in T cells, either in the solid tumor tissue or in the circulating blood. In addition, based on an in vitro co-culture study, a higher tumor burden led to the suppression of the anti-tumor response of T cells. Furthermore, we found that a higher tumor burden was correlated with the differentiation of monocytes to tumor associated macrophages in the tumor micro-environment. Both results demonstrate the importance of considering both the immune system and tumor burden for prognostic analysis. CONCLUSION: Our study has identified a novel clinical biomarker, namely, the immune response to tumor burden ratio, that can be used to distinguish survival outcomes in DLBCL patients, and demonstrated the potential mechanism underlying the use of this biomarker, that incorporates both the immune system and tumor burden, for use in future clinical applications.

Cu x O@DNA sphere-based electrochemical bioassay for sensitive detection of Pb2.

The paper describes a one-step synthetic method to chemically reduce cupric sulfate by ascorbic acid in the presence of DNA strands to directly produce Cu x O@DNA spheres. The DNA strands act as template to assist the preparation of Cu x O, and also are capable of specifically binding  Pb(II) ions. The Cu x O@DNA spheres possess high specific surface area and strong bioaffinity. They can be directly employed as platform for detecting Pb2+ sensitively. Electrochemical impedance spectroscopy data showed that the assay exhibits high sensitivity and a wide linear analytical range that extends from 0.1 to 100 nM, and the detection limit is 6.8 pM at a signal-to-noise ratio of 3. The assay is selective, acceptably reproducible, stable, and well feasible for the detection of Pb2+ in blood serum. Graphical abstract Schematic presentation of the preparation of DNA-templated Cu x O spheres (Cu x O@DNA) for use in electrochemical detection of Pb2+. The assay exhibits detection limit of 6.8 pM, high selectivity, acceptable reproducibility, stability, and good applicability for Pb2+ detection.

FoxP3 in papillary thyroid carcinoma induces NIS repression through activation of the TGF-ß1/Smad signaling pathway.

Forkhead box P3 (FoxP3) expression in papillary thyroid carcinoma (PTC) is associated with resistance to radioiodine treatment. The sodium iodine symporter (NIS) is a plasma membrane glycoprotein, the repression of which may render the tumor refractive to radioiodine therapy. In this study, samples from 90 PTCs as well as 40 normal thyroid tissues were examined for FoxP3 and NIS by immunohistochemistry and real-time PCR. We found that FoxP3 was associated with decreased NIS expression. Lentiviral-mediated FoxP3-overexpressing cells were constructed and real-time PCR and western blotting were performed to evaluate the expression of NIS. Meanwhile, key members of the transforming growth factor-ß1 (TGF-ß1) pathway were explored by ELISA and immunofluorescence and a neutralizing TGF-ß1 antibody was used to block activity. In vitro, FoxP3 overexpression significantly reduced NIS transcript and protein levels and the TGF-ß1 pathway was activated. However, treatment with neutralizing TGF-ß1 antibody partially abrogated FoxP3-induced NIS repression. These findings suggest that FoxP3 could compromise NIS expression by inducing TGF-ß1.

Quantitative assessment of HER2 amplification in HER2-positive breast cancer: its association with clinical outcomes.

Human epidermal growth factor receptor 2 (HER2) is an effective therapeutic target in breast cancer. However, not all patients benefit from trastuzumab-based therapy. We aimed to investigate whether patients with different levels of HER2 amplification would experience different clinical outcomes with trastuzumab-based chemotherapy. We quantified the HER2 gene copy number (GCN) and HER2/centromere chromosome probe 17 (CEP17) ratio in 291 breast cancer patients with HER2 amplification confirmed by immunohistochemistry and fluorescence in situ hybridization. The optimal cutoff points for HER2 GCN and the HER2/CEP17 ratios for distinguishing positive results were determined by receiver operating characteristic (ROC) curve analyses. ROC analysis identified optimal cutoff points for HER2 GCN and HER2/CEP17 ratios as 11.5 and 6.5 (P = 0.039 and P = 0.012), respectively. The DFS in patients with HER2 GCN <11.5 was significantly longer than in HER2 GCN ≥11.5 patients (P = 0.015) according to Kaplan-Meier survival curves analysis. Similarly, patients with HER2/CEP17 ratios <6.5 had a significantly longer DFS than those with HER2/CEP17 ratios ≥6.5 (P = 0.013). Moreover, patients with HER2 cluster amplification showed a worse survival than those with HER2 non-cluster amplification (P = 0.041). This study demonstrated a significant association between the level of HER2 amplification and survival time in a relatively large cohort of HER2-positive breast cancer patients undergoing trastuzumab-based chemotherapy. Further investigations of more precise quantitative measurements and larger cohorts are required to define this threshold.

Increased number of forkhead box P3+ tumor-infiltrating lymphocytes correlates with high preoperative albumin level and better survival in patients with stage II or III colorectal cancer.

Tumor-infiltrating lymphocytes (TILs) that test positive for forkhead box P3 (FOXP3) and elevated preoperative serum albumin levels have been positively associated with survival in colorectal cancer (CRC). This study aimed to investigate correlations among FOXP3+ TILs, preoperative serum albumin, overall survival, and other clinicopathological features of CRC patients. Surgical specimens from 340 stage II-III CRC patients were stained immunohistochemically for the presence of FOXP3+ TILs. Serum albumin levels were determined using an automatic biochemistry analyzer. Associations between various clinicopathological features and patient survival were analyzed via a Cox proportional hazards regression model. The correlation between FOXP3+ TILs and preoperative serum albumin was assessed using Pearson's correlation analysis. Survival curves were constructed by the Kaplan-Meier method. A high FOXP3+ TIL density (>15/five high-power fields), elevated preoperative serum albumin (≥35 g/L), and proximal colon carcinoma were significantly associated with better survival, and high FOXP3+ TIL number and elevated preoperative serum albumin were independent predictors of better survival. The correlation between the number of FOXP3+ TILs and preoperative serum albumin level was significant but neither of these correlated with gender, age, tumor size, tumor differentiation, mucinous tumor, T4 stage, postoperative chemotherapy, or tumor location. Our findings suggest that increased FOXP3+ TILs and high preoperative serum albumin levels are independent prognostic markers for improved survival in CRC patients. Furthermore, the number of FOXP3+ TILs correlates with preoperative serum albumin levels in these patients.

Nesfatin-1 correlates with hypertension in overweight or obese Han Chinese population.

We investigated blood nesfatin-1 levels in hypertension patients. We found that fasting plasma nesfatin-1 levels were significantly higher in hypertension patients than in control groups, especially in overweight/obese hypertension patients (4.5 ± 2.1 versus 3.3 ± 1.1 ng/ml, p < 0.01). Body mass index, systolic blood pressure and diastolic blood pressure were indeed positively correlated with fasting plasma nesfatin-1 levels (r = 0.234, p < 0.05; r = 0.304, p < 0.01; r = 0.251, p < 0.05; r = 0.461, p < 0.01; respectively). Logistic regression analysis revealed that the plasma level of nesfatin-1 could be independent of risk prediction over standard measures (OR = 1.547, 95% CI: 1.153-6.273, p = 0.026). Nesfatin-1 has the incremental contribution to hypertension risk prediction (IDI: 0.014, p = 0.018; NRI: 0.050, p = 0.043). The plasma nesfatin-1 level in hypertension patients with microalbuminuria are significantly higher than those without microalbuminuria patients (6.4 ± 2.1 ng/ml versus 3.9 ± 1.8 ng/ml, p < 0.01). Nesfatin-1 might play an important role in obesity hypertension, and its increase could be a risk factor for obesity-associated hypertension.

The effect of resveratrol on the expression of AdipoR1 in kidneys of diabetic nephropathy.

Adiponectin is an adipocyte derived protein that plays pivotal roles in anti-oxidation, anti-inflammatory and insulin-sensitizing properties by activating two receptors, AdipoR1 and AdipoR2. Recent studies have shown that the down-regulation of AdipoR1 is a known cause of diabetic nephropathy (DN). Resveratrol (Resv), a natural polyphenol, has been identified as a potent activator of forkhead transcription factor O1 (FoxO1) which can up-regulate the expression of AdipoR1. In the present study, we have investigated whether Resv can up-regulate the expression of AdipoR1 by activating FoxO1 that is in kidney of DN rats and mesangial cells (MCs) cultured in high glucose (HG, 30 mmol/L) medium. In vivo, we show that, in the renal cortex of diabetic rats, the expression of AdipoR1 was significantly reduced and correlated with an increase in the generation of malondialdehyde (MDA), Collagen IV and fibronectin proteins. However, administration with Resv significantly increased the expression of AdipoR1. This correlated with not only a decrease in generation of MDA, Collagen IV and fibronectin proteins levels but also more improved kidney pathological and biochemical indicators changes. In vitro, we show that HG-induced depression of FoxO1 activity was associated with the expression of Adipor1 in MCs. Treatment with Resv (20 µmol/L) caused an elevation in the activity of FoxO1 and a significantly increase in the expression of AdipoR1. Furthermore, inhibition of FoxO1 through short hairpin RNA markedly reduced the expression of Adipor1 in MCs cultured by Resv. In conclusion, Resv can significantly increase the expression of AdipoR1 by activating FoxO1 in diabetic kidney. These data also suggest that Resv may serve as a promising agent for preventing or treating DN.

Protective effect of cytosolic phospholipase A2 inhibition against inflammation and degeneration by promoting regulatory T cells in rats with experimental autoimmune encephalomyelitis.

Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme that initiates the production of various inflammatory mediators. Previous studies have shown that inhibiting cPLA2 exerts a neuroprotective effect on experimental autoimmune encephalomyelitis (EAE) by ameliorating the severity of the disease and influencing Th1 and Th17 responses. However, it remains unclear whether treatment with a cPLA2 inhibitor will influence the regulatory T cells (Tregs) that play a critical role in maintaining immune homeostasis and preventing autoimmune diseases. In this study, the cPLA2 inhibitor AX059 reduced the onset and progression of EAE in Lewis rats. In addition, this effect was accompanied by activation of Tregs and alterations in the expression of their various cytokines. The study therefore demonstrated that Tregs are involved in the immunomodulatory effect mediated by cPLA2 inhibition. These findings may have clinical application in the treatment of multiple sclerosis.

Segmentation-free measurement of locomotor frequency in Caenorhabditis elegans using image invariants.

An animal's locomotor rate is an important indicator of its motility. In studies of the nematode C. elegans, assays of the frequency of body bending waves have often been used to discern the effects of mutations, drugs, or aging. Traditional manual methods for measuring locomotor frequency are low in throughput and subject to human error. Most current automated methods depend on image segmentation, which requires high image quality and is prone to errors. Here, we describe an algorithm for automated estimation of C. elegans locomotor frequency using image invariants, i.e., shape-based parameters that are independent of object translation, rotation, and scaling. For each video frame, the method calculates a combination of 8 Hu's moment invariants and a set of Maximally Stable Extremal Regions (MSER) invariants. The algorithm then calculates the locomotor frequency by computing the autocorrelation of the time sequence of the invariant ensemble. Results of our method show excellent agreement with manual or segmentation-based results over a wide range of frequencies. We show that compared to a segmentation-based method that analyzes a worm's shape and a method based on video covariance, our technique is more robust to low image quality and background noise. We demonstrate the system's capabilities by testing the effects of serotonin and serotonin pathway mutations on C. elegans locomotor frequency.

Automated multimodal imaging of Caenorhabditis elegans behavior in multi-well plates.

Large-scale assays of behavior in model organisms play an important role in genetic screens, drug testing, and the elucidation of gene-behavior relationships. We have developed an automated, high-throughput imaging and analysis method for assaying behaviors of the nematode C. elegans . We use high-resolution optical imaging to longitudinally record the behaviors of 96 animals at a time in multi-well plates, and computer vision software to quantify the animals' locomotor activity, behavioral states, and egg laying events. To demonstrate the capabilities of our system we used it to examine the role of serotonin in C. elegans behavior. We found that egg-laying events are preceded by a period of reduced locomotion, and that this decline in movement requires serotonin signaling. In addition, we identified novel roles of serotonin receptors SER-1 and SER-7 in regulating the effects of serotonin on egg laying across roaming, dwelling, and quiescent locomotor states. Our system will be useful for performing genetic or chemical screens for modulators of behavior.

Segmentation-free measurement of locomotor frequency in Caenorhabditis elegans using image invariants.

An animal's locomotor rate is an important indicator of its motility. In studies of the nematode C. elegans, assays of the frequency of body bending waves have often been used to discern the effects of mutations, drugs, or aging. Traditional manual methods for measuring locomotor frequency are low in throughput and subject to human error. Most current automated methods depend on image segmentation, which requires high image quality and is prone to errors. Here, we describe an algorithm for automated estimation of C. elegans locomotor frequency using image invariants, i.e., shape-based parameters that are independent of object translation, rotation, and scaling. For each video frame, the method calculates a combination of 8 Hu's moment invariants and a set of Maximally Stable Extremal Regions (MSER) invariants. The algorithm then calculates the locomotor frequency by computing the autocorrelation of the time sequence of the invariant ensemble. Results of our method show excellent agreement with manual or segmentation-based results over a wide range of frequencies. We show that compared to a segmentation-based method that analyzes a worm's shape and a method based on video covariance, our technique is more robust to low image quality and background noise. We demonstrate the system's capabilities by testing the effects of serotonin and serotonin pathway mutations on C. elegans locomotor frequency.