Getting on the right track: Interactions between viruses and the cytoskeletal motor proteins.

The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments and their accessory proteins. Motor proteins, which comprise the dynein, kinesin and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along with the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as rabies disease and acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, that is, rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins.

Manipulation of selective macroautophagy by pathogens at a glance.

Macroautophagy (hereafter autophagy) is a highly conserved catabolic pathway, which mediates the delivery of unwanted cytoplasmic structures and organelles to lysosomes for degradation. In numerous situations, autophagy is highly selective and exclusively targets specific intracellular components. Selective types of autophagy are a central element of our cell-autonomous innate immunity as they can mediate the turnover of viruses or bacteria, that gain access to the cytoplasm of the cell. Selective autophagy also modulates other aspects of our immunity by turning over specific immunoregulators. Throughout evolution, however, the continuous interaction between this fundamental cellular pathway and pathogens has led several pathogens to develop exquisite mechanisms to inhibit or subvert selective types of autophagy, to promote their intracellular multiplication. This Cell Science at a Glance article and the accompanying poster provides an overview of the selective autophagy of both pathogens, known as xenophagy, and of immunoregulators, and highlights a few archetypal examples that illustrate molecular strategies developed by viruses and bacteria to manipulate selective autophagy for their own benefit.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

What Is Breast in the Bone?

The normal developmental program that prolactin generates in the mammary gland is usurped in the cancerous process and can be used out of its normal cellular context at a site of secondary metastasis. Prolactin is a pleiotropic peptide hormone and cytokine that is secreted from the pituitary gland, as well as from normal and cancerous breast cells. Experimental and epidemiologic data suggest that prolactin is associated with mammary gland development, and also the increased risk of breast tumors and metastatic disease in postmenopausal women. Breast cancer spreads to the bone in approximately 70% of cases with advanced breast cancer. Despite treatment, new bone metastases will still occur in 30%-50% of patients. Only 20% of patients with bone metastases survive five years after the diagnosis of bone metastasis. The breast cancer cells in the bone microenvironment release soluble factors that engage osteoclasts and/or osteoblasts and result in bone breakdown. The breakdown of the bone matrix, in turn, enhances the proliferation of the cancer cells, creating a vicious cycle. Recently, it was shown that prolactin accelerated the breast cancer cell-mediated osteoclast differentiation and bone breakdown by the regulation of breast cancer-secreted proteins. Interestingly, prolactin has the potential to affect multiple proteins that are involved in both breast development and likely bone metastasis, as well. Prolactin has normal bone homeostatic roles and, combined with the natural "recycling" of proteins in different tissues that can be used for breast development and function, or in bone function, increases the impact of prolactin signaling in breast cancer bone metastases. Thus, this review will focus on the role of prolactin in breast development, bone homeostasis and in breast cancer to bone metastases, covering the molecular aspects of the vicious cycle.

Coronavirus entry and release in polarized epithelial cells: a review.

Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population.

The preparation and characterization of micelles from poly(γ-glutamic acid)-graft-poly(L-lactide) and the cellular uptake thereof.

Chemotherapy is a traditional therapeutic approach for the treatment of many solid tumors, but the poor solubility and low bioavailability of hydrophobic anti-cancer drugs greatly limit their applications. In this article, DOX-loaded micelles were fabricated based on an amphiphilic graft polymer composed of hydrophilic poly(γ-glutamic acid) (γ-PGA) and hydrophobic poly (L-lactide) (PLLA). The structure of the copolymers and the characteristic of the micelles were studied. The release profiles of doxorubicin as a model drug from the micelles were measured. Due to the protonation of the amino group of DOX and the conformational alteration of γ-PGA, the release of DOX from γ-PGA-g-PLLA micelle was faster in the acid condition, which is beneficial to tumor therapy. The cellular uptake of the DOX-loaded γ-PGA-g-PLLA micelle was proved to be a GGT-mediated process.

Small-Molecule Allosteric Inhibitors of Human Aspartate Transcarbamoylase Suppress Proliferation of Bone Osteosarcoma Epithelial Cells.

Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120 nM) in an in vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10 µM. Evidence for a druggable allosteric pocket in human ATC is provided by both in vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.

Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus.

The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection.

WDR45, one gene associated with multiple neurodevelopmental disorders.

The WDR45 gene is localized on the X-chromosome and variants in this gene are linked to six different neurodegenerative disorders, i.e., ß-propeller protein associated neurodegeneration, Rett-like syndrome, intellectual disability, and epileptic encephalopathies including developmental and epileptic encephalopathy, early-onset epileptic encephalopathy and West syndrome and potentially also specific malignancies. WDR45/WIPI4 is a WD-repeat ß-propeller protein that belongs to the WIPI (WD repeat domain, phosphoinositide interacting) family. The precise cellular function of WDR45 is still largely unknown, but deletions or conventional variants in WDR45 can lead to macroautophagy/autophagy defects, malfunctioning mitochondria, endoplasmic reticulum stress and unbalanced iron homeostasis, suggesting that this protein functions in one or more pathways regulating directly or indirectly those processes. As a result, the underlying cause of the WDR45-associated disorders remains unknown. In this review, we summarize the current knowledge about the cellular and physiological functions of WDR45 and highlight how genetic variants in its encoding gene may contribute to the pathophysiology of the associated diseases. In particular, we connect clinical manifestations of the disorders with their potential cellular origin of malfunctioning and critically discuss whether it is possible that one of the most prominent shared features, i.e., brain iron accumulation, is the primary cause for those disorders.Abbreviations: ATG/Atg: autophagy related; BPAN: ß-propeller protein associated neurodegeneration; CNS: central nervous system; DEE: developmental and epileptic encephalopathy; EEG: electroencephalograph; ENO2/neuron-specific enolase, enolase 2; EOEE: early-onset epileptic encephalopathy; ER: endoplasmic reticulum; ID: intellectual disability; IDR: intrinsically disordered region; MRI: magnetic resonance imaging; NBIA: neurodegeneration with brain iron accumulation; NCOA4: nuclear receptor coactivator 4; PtdIns3P: phosphatidylinositol-3-phosphate; RLS: Rett-like syndrome; WDR45: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting.

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease.

The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

[Comparison of the therapeutic effects of pleural perfusion of NDP and cDDP in NSCLC patients with malignant pleural effusion].

OBJECTIVE: To compare the therapeutic effects of pleural perfusion of NDP and cDDP in non-small cell lung cancer (NSCLC) patients with malignant pleural effusion, their quality of life and toxic side effects. METHODS: Sixty-eight NSCLC patients with malignant pleural effusion after chest drainage were randomly divided into two groups according to the pathological types: 34 cases in the NDP (Group A) and cDDP groups (Group B), 34 cases each. They were treated with NDP (40 mg/m(2)) and dexamethasone (10 mg) dissolved in 40 ml normal saline, or cDDP (40 mg/m(2)) and dexamethasone (10 mg) dissolved in 40 ml of normal saline, respectively, through pleural perfusion weekly for 2-4 weeks. Routine and symptomatic treatment was used in all the patients. The therapeutic effects, life quality and toxic side effects were evaluated. RESULTS: The response rates of groups A and B were 88.23% and 61.7%, respectively, (P < 0.01). The rates of toxic side effects in groups A and B were 39.6% and 41.9%, respectively, (P > 0.05). However, the rates of gastrointestinal side effects of the two groups were 5% and 12.9%, respectively, (P < 0.05). The Karnofsky scores of group A were higher than that in group B (P < 0.05). The survival time of group A was significantly longer than that of group B. CONCLUSION: Pleural perfusion with NDP is a good treatment method with milder toxicity for patients with malignant pleural effusion caused by NSCLC.

Repurposing the HCV NS3-4A protease drug boceprevir as COVID-19 therapeutics.

The rapid growth of COVID-19 cases is causing an increasing death toll and also paralyzing the world economy. De novo drug discovery takes years to move from idea and/or pre-clinic to market, and it is not a short-term solution for the current SARS-CoV-2 pandemic. Drug repurposing is perhaps the only short-term solution, while vaccination is a middle-term solution. Here, we describe the discovery path of the HCV NS3-4A protease inhibitors boceprevir and telaprevir as SARS-CoV-2 main protease (3CLpro) inhibitors. Based on our hypothesis that α-ketoamide drugs can covalently bind to the active site cysteine of the SARS-CoV-2 3CLpro, we performed docking studies, enzyme inhibition and co-crystal structure analyses and finally established that boceprevir, but not telaprevir, inhibits replication of SARS-CoV-2 and mouse hepatitis virus (MHV), another coronavirus, in cell culture. Based on our studies, the HCV drug boceprevir deserves further attention as a repurposed drug for COVID-19 and potentially other coronaviral infections as well.

The Interaction between Nidovirales and Autophagy Components.

Autophagy is a conserved intracellular catabolic pathway that allows cells to maintain homeostasis through the degradation of deleterious components via specialized double-membrane vesicles called autophagosomes. During the past decades, it has been revealed that numerous pathogens, including viruses, usurp autophagy in order to promote their propagation. Nidovirales are an order of enveloped viruses with large single-stranded positive RNA genomes. Four virus families (Arterividae, Coronaviridae, Mesoniviridae, and Roniviridae) are part of this order, which comprises several human and animal pathogens of medical and veterinary importance. In host cells, Nidovirales induce membrane rearrangements including autophagosome formation. The relevance and putative mechanism of autophagy usurpation, however, remain largely elusive. Here, we review the current knowledge about the possible interplay between Nidovirales and autophagy.

Fully transparent high performance thin film transistors with bilayer ITO/Al-Sn-Zn-O channel structures fabricated on glass substrate.

In this work, fully transparent high performance double-channel indium-tin-oxide/Al-Sn-Zn-O thin-film transistors (ITO/ATZO TFTs) are successfully fabricated on glass by radio frequency (RF) magnetron sputtering. The ITO layer acts as the bottom channel layer to increase the channel carrier concentration. The top ATZO channel layer, which is deposited via high oxygen partial pressure in the sputtering process, is useful to control the minimum off-state current. After annealing, the ITO/ATZO TFT demonstrates outstanding electrical performances, including a high ON/OFF current ratio (Ion/Ioff) of 3.5 × 108, a steep threshold swing (SS) of 142.2 mV/decade, a superior saturation mobility (µsat) of 246.0 cm2/Vs, and a threshold voltage VT of 0.5 V. The operation mechanisms for double-channel structures are also clarified.

Differential expression profiles of long non-coding RNAs as potential biomarkers for the early diagnosis of acute myocardial infarction.

Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. The early diagnosis of AMI is crucial for deciding the course of treatment and saving lives. Long non-coding RNAs (lncRNAs) are recently discovered ncRNA class and their dysregulated expression has been implicated in cardiovascular diseases. In this study, we analyzed lncRNA expression pattern by using two microarray datasets of AMI and healthy samples from the Gene Expression Omnibus (GEO) database and tried to identify novel AMI-related lncRNAs and investigate the predictive roles of lncRNAs in the early diagnosis of AMI. From the discovery cohort, 11 differentially expressed lncRNAs were identified as candidate biomarkers that were validated in the discovery cohort, internal cohort and an independent cohort, respectively. Hierarchical clustering analysis suggested that the expression pattern of these 11 candidate lncRNA biomarkers was closely associated with disease status of samples. Then a lncRNA risk classifier was developed by integrating expression value of 11 differentially expressed lncRNAs using support vector machine (SVM) algorithm. The results of leaving one out cross-validation (LOOCV) suggested that the lncRNA risk classifier has a good discrimination between AMI patients and healthy samples with the area under ROC curve (AUC) of 0.955, 0.92 and 0.701 in three cohorts, respectively. Functional enrichment analysis suggested that these 11 candidate lncRNA biomarkers might be involved in inflammation- and immune-related biological processes. Our study indicates the potential roles in the early diagnosis of AMI and will improve our understanding of the molecular mechanism of the occurrence and recurrence of AMI.

Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers.

Coronaviruses (CoV) are enveloped viruses and rely on their nucleocapsid N protein to incorporate the positive-stranded genomic RNA into the virions. CoV N proteins form oligomers but the mechanism and relevance underlying their multimerization remain to be fully understood. Using in vitro pull-down experiments and density glycerol gradients, we found that at least 3 regions distributed over its entire length mediate the self-interaction of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) N protein. The fact that these regions can bind reciprocally between themselves provides a possible molecular basis for N protein oligomerization. Interestingly, cytoplasmic N molecules of MHV-infected cells constitutively assemble into oligomers through a process that does not require binding to genomic RNA. Based on our data, we propose a model where constitutive N protein oligomerization allows the optimal loading of the genomic viral RNA into a ribonucleoprotein complex via the presentation of multiple viral RNA binding motifs.

Ultrastructural Characterization of Membrane Rearrangements Induced by Porcine Epidemic Diarrhea Virus Infection.

The porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) belonging to the α-CoV genus and it causes high mortality in infected sucking piglets, resulting in substantial losses in the farming industry. CoV trigger a drastic reorganization of host cell membranes to promote their replication and egression, but a detailed description of the intracellular remodeling induced by PEDV is still missing. In this study, we examined qualitatively and quantitatively, using electron microscopy, the intracellular membrane reorganization induced by PEDV over the course of an infection. With our ultrastructural approach, we reveal that, as most of CoV, PEDV initially forms double-membrane vesicles (DMVs) and convoluted membranes (CMs), which probably serve as replication/transcription platforms. Interestingly, we also found that viral particles start to form almost simultaneously in both the endoplasmic reticulum and the large virion-containing vacuoles (LVCVs), which are compartments originating from the Golgi, confirming that α-CoV assemble indistinguishably in two different organelles of the secretory pathway. Moreover, PEDV virons appear to have an immature and a mature form, similar to another α-CoV the transmissible gastroenteritis coronavirus (TGEV). Altogether, our study underlies the similarities and differences between the lifecycle of α-CoV and that of viruses belonging to other CoV subfamilies.

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific real-time RT-PCR, indirect immunofluorescence assays and electron microscopy. In addition, the open reading frame sequence (ORF) of the EMCV C15 virus was determined. From sequence comparison and phylogenetic analysis among 24 reference EMCV strains, it appears that the EMCV C15 strain is closely genetically related to strain BEL2887A/91 (>99.0% nucleotide identity). In artificially challenged dogs, the heart and brain were important targets of EMCV C15. This study provides genetic and pathogenic characterization of the EMCV C15 strain isolated in Beijing and calls for sustained surveillance of EMCV infection in China to support better prevention and control of the disease.

Fully transparent flexible tin-doped zinc oxide thin film transistors fabricated on plastic substrate.

In this work, we have successfully fabricated bottom gate fully transparent tin-doped zinc oxide thin film transistors (TZO TFTs) fabricated on flexible plastic substrate at low temperature by RF magnetron sputtering. The effect of O2/Ar gas flow ratio during channel deposition on the electrical properties of TZO TFTs was investigated, and we found that the O2/Ar gas flow ratio have a great influence on the electrical properties. TZO TFTs on flexible substrate has very nice electrical characteristics with a low off-state current (Ioff) of 3 pA, a high on/off current ratio of 2 × 107, a high saturation mobility (µsat) of 66.7 cm2/V•s, a steep subthreshold slope (SS) of 333 mV/decade and a threshold voltage (Vth) of 1.2 V. Root-Mean-Square (RMS) roughness of TZO thin film is about 0.52 nm. The transmittance of TZO thin film is about 98%. These results highlight that the excellent device performance can be realized in TZO film and TZO TFT can be a promising candidate for flexible displays.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

BACKGROUND: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. METHODS: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. RESULTS: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. CONCLUSIONS: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.