Palmitic acid induces lipid droplet accumulation and senescence in nucleus pulposus cells via ER-stress pathway.

Intervertebral disc degeneration (IDD) is a highly prevalent musculoskeletal disorder affecting millions of adults worldwide, but a poor understanding of its pathogenesis has limited the effectiveness of therapy. In the current study, we integrated untargeted LC/MS metabolomics and magnetic resonance spectroscopy data to investigate metabolic profile alterations during IDD. Combined with validation via a large-cohort analysis, we found excessive lipid droplet accumulation in the nucleus pulposus cells of advanced-stage IDD samples. We also found abnormal palmitic acid (PA) accumulation in IDD nucleus pulposus cells, and PA exposure resulted in lipid droplet accumulation and cell senescence in an endoplasmic reticulum stress-dependent manner. Complementary transcriptome and proteome profiles enabled us to identify solute carrier transporter (SLC) 43A3 involvement in the regulation of the intracellular PA level. SLC43A3 was expressed at low levels and negatively correlated with intracellular lipid content in IDD nucleus pulposus cells. Overexpression of SLC43A3 significantly alleviated PA-induced endoplasmic reticulum stress, lipid droplet accumulation and cell senescence by inhibiting PA uptake. This work provides novel integration analysis-based insight into the metabolic profile alterations in IDD and further reveals new therapeutic targets for IDD treatment.

Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Hepatocellular Carcinoma LINC01116 Outcompetes T Cells for Linoleic Acid and Accelerates Tumor Progression.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a highly immunosuppressive tumor microenvironment and a typical pattern of disturbances in hepatic lipid metabolism. Long non-coding RNAs are shown to play an important role in the regulation of gene expression, but much remains unknown between tumor microenvironment and lipid metabolism as a bridging molecule. Here, long intergenic nonprotein coding RNA 01116 (LINC01116) acts as this molecular which is frequently upregulated in HCC patients and associated with HCC progression in vitro and in vivo is identified. Mechanistically, LINC01116 stabilizes EWS RNA-binding protein 1 (EWSR1) by preventing RAD18 E3 Ubiquitin Protein Ligase (RAD18) -mediated ubiquitination. The enhanced EWSR1 protein upregulates peroxisome proliferator activated receptor alpha (PPARA) and fatty acid binding protein1 (FABP1) expression, a long-chain fatty acid (LCFA) transporter, and thus cancer cells outcompete T cells for LCFAs, especially linoleic acid, for seeding their own growth, leading to T cell malfunction and HCC malignant progression. In a preclinical animal model, the blockade of LINC01116 leads to enhanced efficacy of anti-PD1 treatment accompanied by increased cytotoxic T cell and decreased exhausted T cell infiltration. Collectively, LINC01116 is an immunometabolic lncRNA and the LINC01116-EWSR1-PPARA-FABP1 axis may be targetable for cancer immunotherapy.

Colloidal Synthesis of Hollow Double Perovskite Nanocrystals and Their Applications in X-ray Imaging.

Rare earth-based halide double perovskites are regarded as an emerging class of X-ray scintillation materials. However, the majority of related scintillator applications are still focused on single crystal and powder systems; the application of nanocrystal (NC) scintillators is rarely reported. Here, we present the synthesis of high-purity Cs2NaTbCl6 NCs by an improved hot-injection method. Interestingly, hollow Cs2NaTbCl6 NCs are observed, the monitoring of the growth process indicates that micrometer-sized NaCl is the initial product, and then the NaCl would convert into Cs2NaTbCl6 NCs through the diffusion of Cs+ and Tb3+ into NaCl lattice, and the faster outward diffusion of Na+ results in the formation of hollow NCs. The double perovskite NCs exhibit green light emission, and the photoluminescence intensity can be significantly enhanced through Ce3+ doping. In particular, the Cs2NaTbCl6:5%Ce3+ scintillator exhibits a linear response and a low detection limit of 79.09 nGy/s when exposed to X-rays. Furthermore, a flexible scintillator film for X-ray imaging is prepared by mixing NCs with polymer, showing a high spatial resolution imaging capability of 10 lp/mm. This work provides a new strategy for hollow perovskite NCs and may shed light on the synthesis of related hollow NCs and their applications in X-ray detection.

C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

BACKGROUND: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CONCLUSIONS: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

LncRNA PWRN1 inhibits the progression of hepatocellular carcinoma by activating PKM2 activity.

Hepatocellular carcinoma (HCC) is one of the most prevalent and leading causes of cancer-related mortality worldwide. Long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in cancer development and progression. The lncRNA PWRN1 (PWRN1), acts as a tumor suppressor factor, which is low expressed in some cancers. However, the molecular mechanisms underlying the effects of PWRN1, especially the regulatory relationship with RNA binding protein in HCC remain largely unknown. In the present study, we demonstrated that PWRN1 was significantly down-regulated in HCC and correlated with better prognosis; furthermore, gain-of-function experiments showed that PWRN1 inhibited the proliferation of HCC cells. We further found that PWRN1 up-regulated pyruvate kinase activity and thus hinders the proliferation of HCC in vitro and in vivo. Mechanistically, pyruvate kinase M2 (PKM2) was bound to it and maintained the high activity state of PKM2, thereby hindering PKM2 from entering the nucleus in the form of low-activity dimers, reducing the expression of c-Myc downstream gene LDHA, leading to a decrease in lactate levels, and inhibiting the growth of tumor cells. In addition, PWRN1 was found to inhibit aerobic glycolysis. Finally, TEPP-46, a pyruvate kinase activator, appeared to inhibit HCC proliferation by maintaining tetramer stability and increasing pyruvate kinase activity. Taken together, our results provide new insights into the biology hindering HCC proliferation and indicate that PWRN1 in combination with PKM2 activators might represent a novel therapeutic target for HCC.

Adsorption Behavior of Organoarsenicals over MnFe2O4-Graphene Hybrid Nanocomposite: The Role of Organoarsenic Chemical Structures.

As a kind of emerging contaminant, organoarsenic compounds have drawn wide concern because of their considerable solubilities in water, and the highly toxic inorganic arsenic species formed during their biotic and abiotic degradation in the natural environment. Thus, the effective removal and studying of the adsorption mechanism of organoarsenic compounds are of significant urgency. In this work, MnFe2O4 and MnFe2O4/graphene were prepared through a facile solvothermal method. From the results of the Transmission Electron Microscope (TEM) characterization, it can be found that MnFe2O4 nanoparticles were uniformly distributed on the surface of the graphene. And the specific surface area of the MnFe2O4/graphene was about 146.39 m2 g-1, much higher than that of the MnFe2O4 (86.15 m2 g-1). The interactions between organoarsenic compounds and adsorbents were conducted to study their adsorption behavior and mechanism. The maximum adsorption capacities of MnFe2O4/graphene towards p-arsanilic acid (p-ASA) and roxarsone (ROX) were calculated to be 22.75 and 30.59 mg g-1. Additionally, the ionic strength, negative ions, and humus were introduced to investigate the adsorption performance of organoarsenic compounds. Electrostatic adsorption and surface complexation are the primary adsorption mechanisms on account of X-ray photoelectron spectroscopy (XPS) and the Fourier-transform infrared spectroscopy (FT-IR) analysis. This research extends the knowledge into studying the interaction between organoarsenic species and hybrid nanomaterials in the natural environment.

Spherical Gauss-Laguerre beam propagation in 4D space-time.

In this paper, what we believe to be a novel class of beams, which are referred to as the spherical Gauss-Laguerre beams, are proposed. The beams propagate stably in the anomalous dispersive media, within which the second order derivative with respect to t could be combined with the two-dimensional (2D) Laplacian operator in the transverse direction and forms a three-dimensional (3D) Laplacian operator, which describes the beam propagation in the z direction within the four-dimensional (4D) x-y-z-t space-time. The wave equation is solved by the variable separation method and the analytical expression for the spherical Gauss-Laguerre beams is derived. The beams have a 3D Gaussian field distribution with a variable beam waist with respect to the propagation distance. Unlike any 2D spatial vortex beams, the 3D beams could possess either the spatial vortex or the spatiotemporal optical vortex (STOV) by choosing the vortex plane in the 3D x-y-t space-time. The derived spherical Gauss-Laguerre beam expression in the 4D space-time is verified by the numerical simulations with excellent agreement.

A Simplified Model for Shear Behavior of Mortar Using Biomimetic Carbonate Precipitation.

As a common molecule in biomineralization, L-aspartic acid (L-Asp) has been proven to be able to induce in vitro CaCO3 precipitation, but its application in sand reinforcement has never been studied. In this study, L-Asp was employed in sand reinforcement for the first time through the newly developed biomimetic carbonate precipitation (BCP) technique. Specimens with different number of BCP spray cycles were prepared, and a series of direct shear tests were conducted to investigate the impact of spray number on shear strength, critical displacement, and residual strength. Then a simplified power model for shear stress-displacement behavior was established and calibrated with the measured data. The results show that BCP can significantly improve the shear strength of sand. As the number of spray cycles increases, both the shear strength and residual strength increase, while the critical displacement decreases. Such variations can be described with two sigmoid models and a linear model, respectively. The simplified power model performs well in most cases, especially at higher spray numbers. This study is expected to provide a practical model for the shear behavior of BCP-treated mortar.

Carbon Storage Tanker Lifetime Assessment.

CO2 capture and storage (CCS) is an important strategy to reduce global CO2 emissions. This work presents both cutting-edge carbon storage tanker design, as well as novel reliability method making possible to extract useful information about the lifespan distribution of carbon capture systems from their recorded time history. The method outlined may be applied on more complex sustainable systems that are exposed to environmental stresses throughout the whole period of their planned service life. The latter is of paramount importance at the design stage for complex engineering systems. Novel design for CCS system is discussed and accurate numerical simulation results are used to apply suggested novel reliability methodology. Furthermore, traditional reliability approaches that deal with complex energy systems are not well suited for handling high dimensionality and cross-correlation between various system components of innovative dynamic CO2 storage subsea shuttle tanker. This study has two distinctive key features: the state of art CCS design concept, and the novel general purpose reliability method, recently developed by authors, and particularly suitable for operational safety study of complex energy systems.

Systematically Study the Tensile and Compressive Behaviors of Diamond-like Carbon.

It is important to understand the mechanical properties of diamond-like carbon (DLC) for use not only in frictionand wear-resistant coatings, but also in vibration reduction and damping increase at the layer interfaces. However, the mechanical properties of DLC are influenced by the working temperature and its density, and the applications of DLC as coatings are limited. In this work, we systematically studied the deformation behaviors of DLC under different temperatures and densities using compression and tensile testing of DLC by molecular dynamics (MD) methods. In our simulation results, the values of tensile stress and compressive stress decreased and tensile strain and compressive strain increased as the temperature increased from 300 K to 900 K during both tensile and compressive processes, indicating that the tensile stress and tensile strain depend on the temperature. During the tensile simulation, Young's modulus of DLC models with different densities had a different sensitivity to the increase in temperature, and the DLC model with a high density was more sensitive than that with a low density, which was not seen in the compression process. We conclude that the Csp3-Csp2 transition leads to tensile deformation, while the Csp2-Csp3 transition and relative slip dominate compressive deformation.

Meta-Structure Hull Design with Periodic Layered Phononic Crystals Theory for Wide-Band Low-Frequency Sound Insolation.

The hulls of marine vehicles are generally very effective at attenuating airborne acoustic noise generated by their powertrains. However, conventional hull designs are generally not very effective at attenuating wide-band low-frequency noise. Meta-structure concepts offer an opportunity for the design of laminated hull structures tailored to address this concern. This research proposes a novel meta-structure laminar hull concept using periodic layered Phononic crystals to optimize the sound insolation performance on the air-solid side of the hull structure. The acoustic transmission performance is evaluated using the transfer matrix, the acoustic transmittance, and the tunneling frequencies. The theoretical and numerical models for a proposed thin solid-air sandwiched meta-structure hull indicate ultra-low transmission within a 50-to-800 Hz frequency band and with two predicted sharp tunneling peaks. The corresponding 3D-printed sample experimentally validates the tunneling peaks at 189 Hz and 538 Hz, with 0.38 and 0.56 transmission magnitudes, respectively, with the frequency band between those values showing wide-band mitigation. The simplicity of this meta-structure design provides a convenient way to achieve acoustic band filtering of low frequencies for marine engineering equipment and, accordingly, an effective technique for low-frequency acoustic mitigation.

Two triplet emitting states in one emitter: Near-infrared dual-phosphorescent Au20 nanocluster.

Intrinsic dual-emission (DE) of gold nanoclusters in the near-infrared (NIR) are fascinating for fundamental importance and practical applications, but their synthesis remains a formidable challenge and sophisticated excited-state processes make elucidating DE mechanisms much more arduous. Here, we report an all-alkynyl-protected gold nanocluster, Au20, showing a prolate Au12 tri-octahedral kernel surrounded by two Au2(CZ-PrA)3 dimers, four Au(CZ-PrA)2 monomers, and two CZ-PrA- bridges. Au20 exhibits distinguished photophysical properties including NIR DE at 820 and 940 nm, microsecond radiative relaxation, and 6.26% photoluminescent quantum yield at ambient environment in nondegassed solution. Combining systematic studies on steady/transient spectroscopy and theoretical calculation, we identified two triplet charge transfer (CT) states, ligand-to-kernel and kernel-based CT states as DE origins. Furthermore, this NIR DE exhibits highly independent and sensitive response to surrounding environments, which well coincide with its mechanism. This work not only provides a substantial structure model to understand a distinctive DE mechanism but also motivates the further development of NIR DE materials.

Carbon Quantum Dots Accelerating Surface Charge Transfer of 3D PbBiO2I Microspheres with Enhanced Broad Spectrum Photocatalytic Activity-Development and Mechanism Insight.

The development of a highly efficient, visible-light responsive catalyst for environment purification has been a long-standing exploit, with obstacles to overcome, including inefficient capture of near-infrared photons, undesirable recombination of photo-generated carriers, and insufficient accessible reaction sites. Hence, novel carbon quantum dots (CQDs) modified PbBiO2I photocatalyst were synthesized for the first time through an in-situ ionic liquid-induced method. The bridging function of 1-butyl-3-methylimidazolium iodide ([Bmim]I) guarantees the even dispersion of CQDs around PbBiO2I surface, for synchronically overcoming the above drawbacks and markedly promoting the degradation efficiency of organic contaminants: (i) CQDs decoration harness solar photons in the near-infrared region; (ii) particular delocalized conjugated construction of CQDs strength via the utilization of photo-induced carriers; (iii) π-π interactions increase the contact between catalyst and organic molecules. Benefiting from these distinguished features, the optimized CQDs/PbBiO2I nanocomposite displays significantly enhanced photocatalytic performance towards the elimination of rhodamine B and ciprofloxacin under visible/near-infrared light irradiation. The spin-trapping ESR analysis demonstrates that CQDs modification can boost the concentration of reactive oxygen species (O2•-). Combined with radicals trapping tests, valence-band spectra, and Mott-Schottky results, a possible photocatalytic mechanism is proposed. This work establishes a significant milestone in constructing CQDs-modified, bismuth-based catalysts for solar energy conversion applications.

Highly Effective Hybrid Copper(I) Iodide Cluster Emitter with Negative Thermal Quenched Phosphorescence for X-Ray Imaging.

The low efficiency triplet emission of hybrid copper(I) iodide clusters is a critical obstacle to their further practical optoelectronic application. Herein, we present an efficient hybrid copper(I) iodide cluster emitter (DBA)4 Cu4 I4 , where the cooperation of excited state structure reorganization and the metallophilicity interaction enables ultra-bright triplet yellow-orange emission with a photoluminescence quantum yield over 94.9 %, and the phonon-assisted de-trapping process of exciton induces the negative thermal quenching effect at 80-300 K. We also investigate the potential of this emitter for X-ray imaging. The (DBA)4 Cu4 I4 wafer demonstrates a light yield higher than 104  photons MeV-1 and a high spatial resolution of ≈5.0 lp mm-1 , showing great potential in practical X-ray imaging applications. Our new copper(I) iodide cluster emitter can serve as a model for investigating the thermodynamic mechanism of photoluminescence in hybrid copper(I) halide phosphorescence materials.

Synthesis of RGO/Cu@ FeAl2O4 Composites and Its Applications in Electromagnetic Microwave Absorption Coatings.

In order to satisfy the requirements of wide frequency bands, the lightweight and strong absorption for the electromagnetic wave absorbing materials, a uniform mixture of FeAl2O4 with RGO/Cu (reduction graphene oxide, RGO) was obtained by the mechanical mixing method, and composite coating was obtained by plasma spraying. The addition of RGO/Cu into FeAl2O4 is conducive to improve the dielectric properties and the impedance matching performance of spinel. When the RGO/Cu composite powders are doped by 10 wt.%, the reflection loss at 15 GHz is -16 dB and the absorption bandwidth is 2 GHz, indicating that the composite material has potential application value in the field of high-frequency wave absorption. The research on the electromagnetic wave absorption mechanism shows that its superior wave absorption performance is determined by the synergistic effect of multiple loss mechanisms such as interfacial polarization, dipole relaxation, natural resonance, exchange resonance, and eddy current loss.

Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.

BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

Effect of Nd Element of Mg-Nd Binary Alloy on the Corrosion Resistance in Sulfate-Reducing Bacteria Solution.

In this paper, the corrosion resistances of Mg-Nd binary alloys with various contents of the neodymium (Nd) element in sulfate-reducing bacteria (SRB) were studied. In the SRB medium, the results of weight loss experiments showed that the increase in the Mg12Nd phase in the alloy increased the galvanic corrosion and the corrosion rate. However, when the continuous network distribution of the second phase formed, the corrosion resistance of the alloy improved. The biofilm was formed by the adhesion of the SRB. Meanwhile, the protection from the corrosion improved due to the corrosion products, which prevent the penetration of corrosive ions. On the other hand, the products of biological metabolism accelerated the corrosion of the matrix.

Offshore tethered platform springing response statistics.

This paper demonstrates the validity of the Naess-Gadai method for extrapolating extreme value statistics of second-order Volterra series processes through application on a representative model of a deep water small size tension leg platform (TLP), with specific focus on wave sum frequency effects affecting restrained modes: heave, roll and pitch. The wave loading was estimated from a second order diffraction code WAMIT, and the stochastic TLP structural response in a random sea state was calculated exactly using Volterra series representation of the TLP corner vertical displacement, chosen as a response process. Although the wave loading was assumed to be a second order (non-linear) process, the dynamic system was modelled as a linear damped mass-spring system. Next, the mean up-crossing rate based extrapolation method (Naess-Gaidai method) was applied to calculate response levels at low probability levels. Since exact solution was available via Volterra series representation, both predictions were compared in this study, namely the exact Volterra and the approximate one. The latter gave a consistent way to estimate efficiency and accuracy of Naess-Gaidai extrapolation method. Therefore the main goal of this study was to validate Naess-Gaidai extrapolation method by available analytical-based exact solution. Moreover, this paper highlights limitations of mean up-crossing rate based extrapolation methods for the case of narrow band effects, such as clustering, typically included in the springing type of response.