Several Late Viséan-Serpukhovian coral reefs were identified in Langping, Tianlin. They provided an opportunity to investigate paleo-environments suitable for the development of reef-building communities and the construction of coral reefs in Langping. In this paper, part of the reef-building environmental and the ecological characteristics of coral reefs then were elaborated by analyzing the development settings, palaeogeography, sedimentation of reefs, the response to hydrodynamic conditions of reef-building corals, effects of disturbance and non-reef-building organism on reef communities, and the influence of coral morphology on reef development. It is considered that the sedimentary environment of Langping in Late Viséan-Serpukhovian is suitable for the development of benthic communities. The current appearance of reefs is determined by both coral populations ecological characteristics and reef-building environment. The discovery of these coral reefs was significant for supplementing the evolution sequence of Carboniferous reefs and studying the development rules of the whole Carboniferous reef ecosystem3. The environmental setting of reef-building communities was one of a series of factors affecting the specific appearance and evolution of reefs, which was the foundation in controlling the overall characteristics of reefs, and played a leading role during the process of reef development and evolution6. Various factors affecting the specific appearance of the Mississippian shallow sea reefs were deduced by investigating reef ecological environments in the Langping and the ecological characteristics of the reef communities.Early Carboniferous coral reefs in southern China were identified in the Yaoyunling formation and Du'an Formation in Xiadong village, Longjiangdong Village and Xinzhai Village, Langping Township, Tianlin County, Guangxi Province Fig. . These r8, with regard to temperature, water quality, light, nutrition and oxygen, there are obvious differences in these environmental factors and ecological features among reefs with diverse appearances. These will result in significant diversity among reefs directly or indirectly12. Fang and Hou13 and Gong's research team16 have conducted significant research on many reefs in Langping. The palaeogeographic environment of Langping is favourable for reconstruction of the benthic communities, by combining lithofacies recognition, biological fossil identification, and sedimentary microfacies analysis15. Several of the reefs developed in the study area are rare late Viséan-Serpukhovian framework reefs with characteristics such as biodiversity, construction patterns, continuity, maturity and size that are different from synchronous reefs in other areas16. These characteristics reflect the unique environmental conditions and ecology of biological communities in the study area. Previous studies on the reef in Langping focused on paleontology, sediment and deposition. This paper takes a two-pronged approach, namely environmental factors and biotope characteristics, to explain the factors and influencing mechanisms that shape the specific appearance of these ancient reefs.Although the ecological conditions for reef development are generally similarField observation, identification, description, measurement, and photographing were carried out at four reef outcrops in Langping. On the basis of clearly observing the boundary of each reef, the exposed extension size of the reefs was accurately measured. The biological species and content, sediments were recorded in detail and photos were taken. About 100 representative samples of sediments and corals were collected from the base, center and roof of each reef, and corresponding data were recorded. Subsequently, more than 130 thin sections, 30 polished slabs surfaces and 200 photos were taken in the lab in preparation for microfacies paleoecological analysis. These samples now are stored in Department of Geology, Northeastern University, Shenyang, China.Diphyphyllum and Siphonodendron with obvious vertical changes in the distribution of reef-building coral assemblages. Three successive reef-building coral population or coral assemblage, Diphyphyllum, Siphonodendron–Lithostrotion, and Diphyphyllum–Syringopora, can be clearly identified from bottom to top.The coral reef outcrop in Xiadong Village were exposed in EW direction and developed into large reefs without obvious discontinuity or evidence of multiple growth cycles. The reef was about 260 m in length and 50 m in height. Main builders consist of colonial corals Diphyphyllum constructed the framework for reef layers. Coral clusters were presented on various scales with maximum of 2 m × 3 m in height and width, most being vertical clusters.The multi-layer reef in Longjiangdong Village could be divided into three layers, 27 m × 3.8 m, 16 m × 2.5 m, and 40 m × 9 m in length and thickness respectively extending nearly EW. Diphyphyllum, solitary Caninia, and colonial Lithostrotionella. The monomeric Tetracoralla eventually formed an upper layer of the reef 3–5 m wide.The patch reef in Longjiangdong Village was smaller, with the outcrop 11.2 m long and 6.2 m thick. The reef builders were colonial Lonsdaleia formed a framework at the base of the reef, overlaid by unidentified dendrimers and colonial coral Antheria. Solitary corals eventually formed the reef’s upper layer, with the thickness of 3–5 m.The coral layer reef preserved in the east of Xinzhai Village was about 15 m long and 5–6 m thick. Colonial Notwithstanding constraints on the amount of hard data, according to our integrated analysis, the developmental environment and ecology of reef communities have an important impact on the appearance of reefs.17. The Carboniferous is generally considered to be a sub-optimal period for the development of framed reefs. After the biological mass extinction, microorganisms and algae rebuilt new reef-building ecosystems19. Some short-term biological frame reefs developed with low diversity, limited reef-building organisms, small sizes, and restricted distribution20. Harsh climate and marine conditions occurred in the Mississippian, including extensive marine hypoxia, repeated glacial and interglacial climate changes, and frequent changes of sea level and seawater surface temperature, potentially hindering the recovery of Early Carboniferous metazoan reefs21.The F/F extinction event in Late Devonian caused the complete recession of the reef-building communities based on stromatoporoid-coral assemblages3 and large-scale reefs could also be seen in some areas although most of the Viséan metazoan reefs were tabular or laminar. Thus, the metazoan skeletal reefs in the middle to late Viséan were considered to have been resurrected due to relatively warm climatic conditions and higher sea levels after a period of complete disintegration at the end of the Devonian and recession at the beginning of the Carboniferous7.Metazoans gradually began to participate in reef building in Early Viséan. A large number of biogenic structures formed by corals and bryozoans began to appear, including a small number of sponge reefs/mounds in the middle and late stage of Viséan. The richness and biodiversity of the Mississippian post-zoobenthic reefs flourished in the late Viséan during which corals, bryozoans, sponges, calcareous microorganisms, and some calcareous algae became the main builders7. Thus, it is expected that more synchronous reefs would to be identified in southern China, or even in the study area in the future.Consequently, the coral reefs in the study area were the products of shallow benthic communities thriving in relatively favourable conditions of Late Viséan-Serpukhovian, which was common for reef development at that time22 regionally  separated from a large platform 28 based on sedimentary characteristics. It suggests that the study area was far away from the margin of steep-slope platform that directly affected by waves, and more consistent with less energetic internal environments of gentle-slope platform.In situ development of mud-crystalline tuffs and muddy tuffs with weak hydrodynamic conditions is common in the Langping area, and evaporites are poorly developed. There were patch reefs and reef layers in different sizes in the wide intraclast beach, where obviously developed reef beach complexes were rare. The fragments of carbonate base broken by storm in the clastic beach haven’t been observed. The study area is considered to be gentle-slope open platform29.On the vast platform of Langping gentle slop, deep water lead to low water energy. While in the coastal area, the water energy was relatively strong, thus coarse-grained bioclastic beach and a small amount of point reef could be developed. The beaches were irregular-shaped due to long term transportation and reformation effects of waves and water flow, showing low and gentle slope angles. Dispersed reef-beach complexes at the platform margin slightly impacted inner-platform seawater and the water flows smoothly30 due to insufficient cohesive and consolidating organisms. In addition, the circumferential impact of water in extensive terraces leads to mud-lime deposition, which is detrimental to most benthic organisms. However, bondstone was more likely to be formed by some binding algaes in the platform (Fig. Therefore, it can be assumed that Langping reefs developed along the intertidal shallows of the terrace. The seawater around Langping carbonate platform in Late Viséan-Serpukhovian was relatively shallow while the water flow was strong. Remains of crinoids, brachiopods, a few foraminifera, and solitary corals were likely broken by strong currents, and deposited in situ with a small amount of gravels and lime-mud Fig. . The claorm Fig. . Therefo28. They were essentially different from the framework coral reefs which developed on steep-slope platform margin that reflected changed hydrodynamic conditions, nutrient sources, reef sizes, and growth rates.At the same time, the warm climate of the late Viséan-Serpukhovian, the good circulation of seawater around the Langping platform, and the abundant supply of oxygen and nutrients were a series of favourable conditions that facilitated the growth of reef-building corals, which led to uplifts being formed on clastic beach, including patch reefs and reef layers with certain sizes. These uplifts impeded waves and provided a protected nearshore environment, though they were much smaller than those developed at the steep-slope platform margin. The inhabitants on the beach could not resist strong waves. These rises were therefore known as reef-beach complexes and could only persist where waves and currents were mild25. The frequent fault activities in Dian-Qian-Gui Basin caused the rise and fall of equivalent sea level. More influence of sea-level fluctuations and hydrodynamic conditions would be exerted on Langping platform due to its small size. Furthermore, reef growth promoted by reef-building communities would be frequently disturbed. The sediments displaying evidence of multicycle sedimentation, different components, and diversely fragile clasts in the study area provided direct evidence of frequently changing environment.Another potentially favourable factor in the study area could be the deeper water area in the gentle-slope sedimentary environment, which could provide more stable conditions and reduce the damaging effects of global glacial events and large scale sea level fluctuations on reef-building communitiesAlternatively, the sedimentary environment of Langping platform provided conditions favorable for reef-building communities to develop and reefs to grow rapidly. These factors directly or indirectly determined the ecology of reef-building communities and the general appearance of reef development in the study area.Overall, the environmental factor is the primary factor affecting the overall development trend of reefs.31. How these ancient reef-building corals adapted to hydrodynamic conditions was reconstructed combining the evolution of reef-building communities with the study.Hydrodynamic conditions are very important factors for reef development, which directly determine the abundance and distribution of each reef-building population and are key factors influencing sedimentation and reef growth, and was particularly evident at Langping. Evidence from the fossils suggested the reef-building corals were also changed in response Table . The hydDiphyphyllum on the bioclastic hard substrates33. They grew vertically into upright clusters (Fig. 34. The relatively dense clumped Siphonodendron and massive Lithostrotion (Fig. Diphyphyllum subordinate with the continuous increase of the water energy, as indicated by the characteristics of sediment particles from fine to coarse. After flourishing for a period, the Siphonodendron–Lithostrotion assemblage eventually waned, likely due to the failure to adapt to the increasing hydrodynamic conditions. Diphyphyllum had persisted combined with Syringopora, to maintain the growth of the reef. However, this assemblage subsequently declined as a result of strong hydrodynamic conditions and finally died out in response to continuous falling of sea level. Consequently, the reefs stopped developing.The Xiadong coral reef started with colonization and expansion of ers Fig. A and werion Fig. B were beDiphyphyllum contributing greatly to reef growth. Diphyphyllum clusters colonized in patchy form on substrates composed of bioclasts or lithic gravels (Fig. Diphyphyllum once again flourished. Diphyphyllum clumps in the unit grew closely together in strong currents, with larger and more sparse individuals than in the lower units. A relatively low energy environment was formed between the Diphyphyllum clusters (Fig. Diphyphyllum could only grow in a limited area of suitability due to the disturbance of high-energy water brought about by short-term sea-level rise and fall. Afterwards, the environment became more favourable and Diphyphyllum expanded rapidly. As a result, the upper unit of Longjiangdong coral reef was formed, in which Diphyphyllum individuals were slightly larger than those in the first two units. Finally, because the kinetic energy of the water continued to weaken, the plaster deposition forced the whole coral reef to stop growing (Fig. The Longjiangdong multi-layer reef was composed of three relatively independent, flat reef layers, suggesting three distinct periods of reef development. Diverse species were identified in the reef, with colonial coral els Fig. A. The fiels Fig. B. Subseqers Fig. C. Subseqing Fig. D.Figure Diphyphyllum initially colonized and expanded in favorable conditions with the increase of water energy. Then the reef-builders transitioned from a single coral species to an assemblage of Diphyphyllum–Caninia–Lithostrotionella. These three coral species grew independently and contribute almost equally to the structure of the reef. However, the structure and function of the coral community were not yet stable enough. It was easily influenced by the weakening hydrodynamics and the increasing sedimentation, resulting in only small patch reefs.Longjiangdong patch reef started to develop in a relatively deep water environment. Lonsdaleia on bioclastic beach. Large coral clusters were formed in the presence of turbulent water. With the weakening of hydrodynamic conditions, an unknown branchlike organism and Antheria communities continued to develop separately in this area. Slender branchlike organisms expanded rapidly in these low-energy water environments until they were replaced by some individual corals as hydrodynamic energy increased. Each builder was short-lived in this layer reef, departing from the reef just at the beginning of colonization and expansion, due to rapidly changed hydrodynamic conditions.The Xinzhai layer reef was initialized by colonization and expansion of The evolution of reef-building corals in these four reefs indicated that both the coral assemblages and coral individuals would constantly adapt to the changing hydrodynamic conditions in Langping as sea level rose and fell. Although this was a reactive adjustment of coral populations in response to long-term environmental impacts, it was clearly positive for the building and development of coral reefs.35. The major disturbances evident in these Mississippian framework reefs were associated with frequent changes of water flow, and drastic changes of climate and weather. These seem to be most obvious in the Langping platform due to its small size, with more frequent environmental influence evident on the reef communities in the study area.Disturbance is a relatively discontinuous event, which is ubiquitous in nature. It may indirectly affect the composition and population structure of reef communities by changing the environmental conditions, thus affect the structure and function of reef communities, even the evolution of the reef36. The conditions for the emergence of ‘Competitive lottery’ also include the need for species in a community to have similar abilities to invade discontinuities and to tolerate environmental conditions.The most direct effect of disturbance events on coral reefs is the disruption of continuously evolving reef communities, which is common in coral reef studies. After the interruption caused by disturbances, some communities gradually recover due to the absence of continuous disturbance, or the dominant biota may be substituted by invading communities. The winner after interruption is decided by random factors to a large extent, in a ‘Competitive lottery’37. In environmental conditions with moderate intensity of disturbance, most species will not disappear entirely. The dominant pioneer species will also be restrained by disturbance to a certain extent, so large number of species can coexist, attaining the highest diversity35.Certainly, low-intensity disturbance does not necessarily produce discontinuity, but medium-intensity disturbance without discontinuity could directly impact on community species diversity. According to the ‘Moderate disturbance hypothesis’, moderate disturbance is conducive to a higher level of community diversity10, which also developed in Dian-Qian-Gui Basin. More than 4 reef-building corals are identified in Xiadong reef, while 4 and 3 are in Xinzhai layer reef, Longjiangdong patch reef respectively. These reef-building corals, mostly Diphyphyllum, Lithostrotion, Siphonodendron and Lonsdaleia, were distributed irregularly in the reefs. Their ecological niche and function were likely similar and none of them was obviously dominant in the community (Fig. The reef-builders in Langping are diverse compared with the Late Carboniferous reefs in Ziyun Countyity Fig. . This is38. It is generally accepted that communities with high diversity are always more stable although ecosystem stability is not absolutely correlated to biodiversity35.The stability of a classical reef ecosystem includes the ability to withstand external disturbances and the ability to return to its original state once the disturbance is removedIvanovia cf. manchurica patch reefs in Ziyun County10, Guizhou province, the different coral assemblages in Langping area could effectively adapt to changing hydrodynamic conditions and maintain reef growth.There have been no reef-building corals with strong resistance and rapid recovery ability in the communities in Langping. None of these corals succeeded in developing into dominant species that can build reef shelves, which made the reefs in Langping mostly appear in the form of small patch reefs or reef layers. However, formation of the large reef in Xiadong Village, patch reef in Longjiangdong Village, and layer reef in Xinzhai Village were all related to their relatively high diversity of reef-building corals. Compared with the situation where only one reef-building organism dominated the Bianping large coral reef, Wengdao large phylloid algal reef and Species diversity increased by disturbance stabilized the ecosystemas shown during the construction of coral reefs in Langping.39. Off-reef organisms are not part of the reef-building community, but also play an important role in participating in energy flow and providing organic matter to the reef ecosystem40.Besides reef-building corals, there were a large number of reef-dwellers and off-reef organisms in the study area. Reef-dwellers referred to the species that didn’t directly contribute to reef growth in the community, mainly including various benthos and algaesCommon reef-dwelling organisms include crinoids, brachiopods, gastropods, various algae, foraminifera, bryozoans and individual corals. Crinoids were overwhelmingly dominant in numbers in the reef samples studied here.46. The large number of crinoids in Langping excluded other metazoans and restricted the development of benthic reef-builders in Late Viséan-Serpukhovian in Langping, leading to poorly developed reef-building communities.Carboniferous echinodermata in Guangxi Province reached its peak in Middle-to-Late Mississippian. In terms of amount and distribution, thick limestone with echinodermata debris in the carbonate platform were often dominated by crinoids47 (Fig. 49. The other was to create a good condition for zoobenthos larvae to dwell and develop, thereby improving species diversity50. The limited productivity of algae in Langping constrained coral reef trophic inputs, which may then have limited populations of dependent metazoans. As a result, algaes and other metazoans were unable to achieve a variety of reef-building patterns, such as bonding, bounding, entanglement52. The reef framework in the study area was not stable in the presence of strong water flow, and the biological communities could not deal with frequent environmental changes, which were directly related to poor development of calcareous algae.Microorganisms and algaes had limited success in stablishing on the moving clastic beach in frequently disturbed water. There has not been obvious evidence of extensive “algal turf” in the coastal area of Langping platform. Only a few corals bonded by algal mats were observed47 Fig. . In addiThe accumulation of reef structure had obvious impact on communities. Large reef structures could support abundance and diverse biota by modifying local environments and creating diverse conditions. Consequently the reef-building communities thrived between disturbances, stabilizing reef construction. In terms of large reef, the framework-building corals would play a key role in reef construction regardless of which kind of patterns was adopted. Therefore, reef-building corals with large size, rapid growth vertically, and strong resistance would become the biggest contributors to reef frame construction.Diphyphyllum, Lithostrotion, Siphonodendron, and Lonsdaleia, etc., being the dominant builders. These corals were similar in morphology such as cluster colony, thick and strong skeleton, and densely packed individuals (Fig. The main reef-building corals in Langping were composed of als Fig. , which eThe complex and diverse local environments formed by large coral reefs can significantly increase benthic populations and improve reef species diversity. As a result, the nutrient flow in the community becomes complicated, and nutrients could be recycled effectively by reducing loss caused by water flow. Therefore, the overall productivity of large coral reef communities was always high. Complex trophic structure satisfied most of the benthos in the community with sufficient nutrients and inorganic salts.The morphology of reef-building corals in Langping enabled them to become predominant species in various water environments, which promoted the continued domed growth of coral reefs and facilitates the development of reef-building communities that form a variety of reefs. It suggests that the morphology of reef-building corals was a key prerequisite for reef development.In conclusion, coral reef communities are always constrained and influenced by environmental conditions. However, the ecology of the inhabitants is also an important factor in the formation of coral reefs.The climate, sea-level conditions in Late Viséan-Serpukhovian and the tropical ocean current flowing through the Langping area ensured that the reef-building corals could continue to survive and build reefs under unfavorable geological background.The gentle slope of Langping platform provided the necessary conditions for the benthic communities. And water environmental factors determined the general appearance of reefs in the study area.The adaptability of the morphological characteristics of reef-building corals to the variable hydrodynamic conditions is of positive significance to the continuous construction of coral reefs.The construction process of reefs has confirmed that disturbance can increase the species diversity of reef communities and enhance the stability of ecosystems, and other biological factors also have a certain impact on the development of reefs.Based on the environment and ecology analysis of the four Late-Mississippian reefs in Langping, new understanding of the influencing factors and evolutionary mechanism of reef-building process are summarized as follows.


The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi–pi/cation–pi interactions. The set of antivirals included fleximers, 5′-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5′-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS. Despite the recent progress in managing coronavirus infectious disease 2019 (COVID-19) and the largely successful vaccination programs, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global threat, and the development of anti-SARS-CoV-2 therapeutics is ongoing. With regards to new druggable targets, increasing attention is being paid to intracellular liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA (gRNA). Growing evidence supports the importance of N-gRNA biomolecular condensates (droplets) formed in the host cell cytoplasm , immune N protein contains two structured domains, namely the N-terminal RNA binding domain (RBD1) and the C-terminal dimerization domain (DD), flanked by three intrinsically disordered regions (IDRs) a. As wasThe dsRNA stretches are enriched in the ORF1ab-harboring 5′-part of SARS-CoV-2 gRNA and can interact with N RBD2. Such interactions supposedly disrupt N dimers and trigger LLPS by exposing the hydrophobic surface of the DD . The preThe spectrum of N-mediated processes suggests the importance of timely N accumulation and phase separation. Interference with N-RNA phase behavior is expected to repress RNA replication and virion assembly . Perhaps the best-known native modulator of SARS-CoV-2 LLPS is ATP. Prevalent in host cells, it controls multiple phase transitions in norm and pathology . Due to In this study, we investigated the LLPS-modulating potential of the recently reported nucleoside/nucleotide-based antivirals. Our ultimate goal was to clarify whether the interference of such small molecules with N-gRNA separation may contribute to the inhibition of SARS-CoV-2 replication. We also aimed to elucidate the general features of potent LLPS modulators. For that, we compared nucleoside/nucleotide-based antivirals containing hydrophobic and polar/negatively charged fragments with perylene-based antivirals containing hydrophobic (polyaromatic) and polar/positively charged fragments. To test the propensity of these compounds for modulating N-gRNA separation, we obtained a simple in vitro model of SARS-CoV-2 condensates c based o2 coverslip with a total area equal to 38 ± 10 µm2) appeared at N concentration equal to 3 µM after overnight incubation (droplet/Fsolution) was equal to 9 ± 6. For subsequent experiments with RNA, we selected N concentration equal to 3 µM, because it has been used in previous studies of N-gRNA LLPS [We aimed to obtain N-RNA droplets that would be a reasonably adequate model of the intracellular condensates ,4  ,8. We usFirst, we fixed N concentration and added increasing concentrations of SL5_long, SL5_short, or pseudoknot RNA b,c. The Next, we focused on SL5_long and SL5_short and performed an additional series of experiments to verify their relative potency at N:RNA ratio equal to 1.5:1 d,e. We uFor further analysis, we selected the SL5_long:N ratio equal to 2:1 (6 µM RNA and 3 µM N) because it ensured noticeable droplet formation, while PC remained equal to 10 ± 3. This N:RNA ratio (1 N protein molecule per ~80 nt) is lower than that used previously (~3 N molecules per nt ) but verBecause SL5_long proved to be the most potent LLPS facilitator, we used it in all subsequent experiments. To confirm the inclusion of SL5_long into the condensates, we obtained its analog with a FAM-labeled stem (tr-SL5_long/SL5-tag) and mixed it 2:1 with N. Following the overnight incubation at 37 °C, droplets similar to those detected in SL5_long:N mixtures b were obAccording to a recent hypothesis, the effects of RNA on N separation can be explained within the framework of the conventional surface tension-related hydrophobicity-driven LLPS concept . BrieflyFirst, we verified the temperature dependence of N-RNA condensates. The exposure of the hydrophobic DD core must be favored by its thermal denaturation . BecauseNext, we analyzed the effects of salt concentration and pH. Both homotypic (N-N) and heterotypic (N-RNA) interactions must be dependent on the ionic strength of the solution. Low ionic strength supposedly facilitates contacts between the negatively charged RNA backbone and the positively charged N stickers. At the same time, it may enhance RNA-RNA repulsion and weaken all hydrophobic interactions. We observed no apparent LLPS at low (10 mM) salt concentrations; the fluorescence microscopy images of the N-RNA mixture were similar to those obtained in the absence of RNA a. This rAt a physiological salt concentration (150 mM) and pH values in the range of 5–7.4, N-RNA mixtures showed increasing LLPS with decreasing pH. In the weakly acidic media, the condensates became apparent even after a relatively short (2 h) incubation at both 20 °C and 37 °C b. IncubaFinally, we verified the effects of the commonly used LLPS modulators 1,6-hexanediol (HD) and ATP. Condensates held together by hydrophobic interactions are typically sensitive to HD . Under pTo summarize this part, N-SL5_long condensates appear to be a reasonably accurate simplified model of the viral condensates because they show appropriate dependence on the external conditions and sensitivity to the known modulators. The condensates are likely held together by both electrostatic and hydrophobic interactions, but hydrophobicity appears to play the key role.The proposed model of the SARS-CoV-2 N-RNA condensates was used to search for LLPS modulators among known antiviral agents and their derivatives . InspireFleximers b are pur4-hydroxycytidine, In addition to purine nucleoside derivatives, we considered a well-known cytidine analog NHC  under physiological conditions  were analyzed using fluorescent microscopy imaging , and thedroplet/Fsolution = 10 ± 4) changed insignificantly (within error) with increasing droplet number (count). S showed a clear linear correlation with droplet count in the cases of ATP, NHC, NorC, and Flex nucleoside derivatives (R2 = 0.83). The correlation was less apparent for non-nucleoside perylene derivatives (R2 = 0.63), probably because they promoted the formation of larger droplets . Within each small-molecule series , droplet size distribution was broad but constant. Thus, we selected S as a key indicator of LLPS efficiency.Under the selected conditions, S did not exceed 5% of the coverslip area in most cases, and the apparent partitioning coefficient . The 5′-norcarbocyclic nucleoside derivatives NorC-24p and NorC-24f were robust facilitators of LLPS. They increased average S values ~11-fold, and ~15-fold, respectively. Top facilitators of LLPS were found in the Peryl series: the hydrophobic non-nucleoside derivative Peryl-2a increased S ~25-fold, and the nucleoside analog Peryl-5 increased it ~50-fold.The hydrophobic perylene derivatives were more potent than amphiphiles, and the derivatives with an extended flexible linker (propyl) between the polyaromatic system and the morpholino ring (Peryl-2b and Peryl-3b) were superior to their shorter-linker (ethyl) analogs (Peryl-2a and Peryl-3a). Importantly, all perylene derivatives induced a more or less pronounced N-RNA gelation and the formation of irregular-shaped aggregates. This occurred during droplet maturation and involved RNA, which we confirmed using tr-SL5_long/SL5tag . In contTo summarize this part, NorC nucleoside analogs and hydrophobic perylene derivatives proved to be top modulators of N-RNA separation. Both of these groups of small molecules enhanced LLPS.Screening of nucleoside analogs and perylene derivatives provided insufficient data for identifying characteristic features of potent LLPS inhibitors but revealed a possible general motif of LLPS enhancers, namely a nucleoside(-like) moiety with an aromatic substituent. Although previously discussed LLPS-related strategies for antiviral drug design rely on LLPS inhibitors ,8,15, enAs evident from Fleximers can also undergo phosphorylation in cells and may trigger mutagenesis or inhibit RNA-dependent RNA polymerase (RdRp). Their activity against SARS-CoV-2 has not been verified so far. We tested Flex-nt10  for RdRp inhibition using a previously developed assay  Supplem and obseIn contrast to mutagens like NHC, close nucleoside mimics, sterically hindered replication terminators, or other RdRp substrates , 5′-norcthe N-terminal RNA binding domain , which supposedly accounts for the specific heterotypic interactions [the C-terminal dimerization domain , which accounts for specific homotypic and various non-specific interactions ;Finally, to clarify the opposite effects of NorC-24p (phase separation enhancer) and the control molecule ATP (phase separation inhibitor) in the LLPS modulation assays, we modeled interactions of these compounds with N protein in silico. NorC-24p and ATP were docked to N domains that reportedly form multiple homotypic (N-N) or heterotypic (N-RNA) contacts upon phase separation a:the N-tDocking revealed different binding sites of ATP and NorC-24p in each N fragment . A compaUsing recombinant N-protein (N) and the YRRRY-rich fragment of the SARS-CoV-2 genome with the extended dsRNA stretch , we obtaBecause N-RNA LLPS is repressed by HD and the excess of ATP according to our data  and prevThe lead perylene derivative Peryl-5 increased condensate formation ~50-fold in our model system and can be used as a robust LLPS modulator in other cell-free systems. However, in cells, it likely targets lipid membranes rather than N-RNA condensates. In contrast, LLPS modulation by 5′-norcarbocyclic derivatives may partly account for their antiviral activity. In silico analysis of the 5′-norcarbocyclic derivative NorC-24p  suggestsRNA of the SARS-CoV-2 Wuhan variant was isolated from a COVID-positive donor’s sample using TRIzol LS Reagent , and reverse transcription of the full-length N protein-encoding fragment was performed using the RevertAid RT Reverse Transcription Kit (Thermo Fisher). The cDNA was PCR-amplified and purified by preparative agarose gel electrophoresis, then digested with BamHI and SalI endonucleases and ligated into the modified  pET22b plasmid that was digested with the same enzymes. The correct assembly of the resulting plasmid pET-CoV2-gN(H), encoding C-His6-tagged N protein, was confirmed by Sanger sequencing.E. coli BL21-gold(DE3) cells were transformed with the pET-CoV2-gN(H) plasmid. A single colony was inoculated into 50 mL of LB medium containing ampicillin (150 lg/mL) and grown in a shaking incubator for 8 h at 30 °C. Following the incubation, the culture was inoculated into 1 L of the fresh LB medium (100 lg/mL ampicillin) and cultivated in a shaker-incubator at 37 °C to an OD600 of ~0.8. Expression was induced by adding IPTG to a final concentration of 0.5 mM. The cells were cultured for an additional 4 h at 37 °C. After incubation, the cells were spun down and disrupted by sonication. The lysate was clarified by centrifugation and filtration. Recombinant C-His6-tagged N-protein was purified by metal chelate affinity chromatography (purity: ≥90%) and labeled (1:1) with a far-red-emitting RED dye using the RED-NHS 2nd Generation Protein Labeling Kit (Nanotemper).Three hairpin-forming modified fragments of SARS-CoV-2 gRNA, Wuhan variant, were obtained by in vitro transcription from T7 promoter-containing dsDNA templates that had been assembled from synthetic oligonucleotides and confirmed by Sanger sequencing. SL5_long [SL5-long :TAATACGACTCACTATAGGGAGAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGACAGTAATTAGTSL5-short :TAATACGACTCACTATAGGGAGAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTTGCATAAGATGGAGAGCCTTGTCCCTGGTTTCAACGACAGTAATTAGTPseudoknot :TAATACGACTCACTATAGGGAGAGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGATReverse transcription was performed using a 2 μg DNA template and HiScribe™ T7 High Yield RNA Synthesis Kit  following the manufacturer’s protocol. Then, the reaction mixture was diluted with a DNAse I buffer and treated with RNAse-free DNAse I at 37 °C to remove the template. RNA was precipitated from cold ethanol, and its purity was verified by electrophoresis in 2% agarose.m > 60 °C) to sustain physiological conditions.FAM label was introduced at the 3′-RNA terminus via the hybrid DNA-RNA duplex tag. For that, a truncated version of the target hairpin (tr-SL5_long) was obtained as described above and hybridized with the synthetic FAM-labeled DNA complement (SL5tag). The resulting duplex has substantial thermal stability :TAATACGACTCACTATAGGGAGAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTSL5-tag (ssDNA):CAGTAATTAGT-FAMAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAAll RNA and RNA/DNA concentrations were measured using NanoDrop 2000 (Thermo Fisher). Secondary structures of the SL5_long, tr-SL5_long/SL5-tag, SL5_short, and pseudoknot RNA were verified by circular dichroism (CD) spectroscopy. The CD spectra of 0.7 µM RNA solutions in the working buffer  were registered at room temperature using a Chirascan spectrophotometer (Applied Photophysics) and a 1 cm optical path quartz cuvette. To visualize the difference between folded  and unfolded (mostly unfolded) RNA, we additionally analyzed SL5_long at 80 °C. To select an appropriate annealing procedure, we compared CD spectra of SL5_long after slow annealing, which supposedly yields the thermodynamically favorable structure but allows for the undesired intermolecular folding, and rapid annealing, which supposedly facilitates intramolecular folding. Rapid annealing was performed as follows: the sample was incubated at 90 °C for 5 min and then snap-cooled on ice. Slow annealing was performed as follows: the sample was incubated at 90 °C for 5 min and then cooled gradually (over 2.5–3 h) to room temperature. For tr-SL5_long/SL5-tag, we also considered a two-step procedure. First, tr-SL5_long was annealed rapidly to enable stem-loop formation. Then, SL5-tag was added, and the mixture was incubated for 2 h at +4 °C to enable duplex formation. The final annealing procedures were selected for each RNA based on the CD data. Because the highest amplitude of the A-form-specific CD band  was obtaTo obtain N-RNA condensates, fluorescently labeled recombinant N-protein (3–9 μM) was mixed with RNA (1–10 μM) in the RNAse-free 20 mM Tris-HCl buffer, pH 7.4, or the 20 mM sodium acetate buffer, pH 5 or 6  and incubated at 20 °C or 37 °C for 2 h or overnight prior to fluorescent microscopy imaging or turbidimetry assays. To evaluate the effects of the small-molecule antivirals on the formation of the condensates, stock solutions of the antivirals in the working buffer or DMSO were added to freshly prepared N-RNA mixtures to a final concentration of 5–20 μM .For turbidimetry assays, all mixtures were prepared in 384-well plates , and optical density at 350–600 nm was registered using M200 Tecan plate reader .For fluorescence microscopy imaging, 3 μL of the N-RNA mixture was placed between glass slides and analyzed using a Nikon Eclipse Ti2 microscope . The droplets were inspected visually for floating. To bona fide discriminate liquid condensates from flocculating aggregates and solid particles, several images of the same area were taken with a time lag of several seconds and compared.droplet/Fsolution = Cdroplet/Csolution, where F is fluorescence intensity and C is concentration) and the average droplet area normalized by the coverslip area . N distribution between droplets and solution was estimated based on Equation (1), which is valid provided the total amount of the protein  remains constant (no precipitation):Using the ImageJ 1.53a software, we calculated the partitioning coefficient  can be rewritten as follows:All experiments were performed in two independent repeats, and in each case at least three large-field (square millimeter) scans were analyzed. Data are presented as means ± SD. The significance of the difference of the mean droplet numbers or S values or between two samples was verified using a standard two-tailed Student’s test.The inhibitory activity of nucleoside analogs and the control compound (NHC) against the SARS-CoV-2 strain PIK35 was verified as was described previously ,31,37. Bhttps://gaussian.com/, accessed on 2 December 2022).3D Models of the ATP and NorC-24 were built using ICM-Pro 3.9 2 . Partial3D models of the N RNA-binding domain, dimerization domain, and the partly disordered central region (aa 233–266) were taken from PDB . Docking was performed using ICM-Pro 3.8.6. The small molecules were rendered flexible, and the protein was fixed. Prior to the docking procedure, the structures of the protein and the small molecules were converted into an ICM object. The parameters needed for interatomic energy calculation and the partial charges for the atoms of the target were taken from the force field ECEPP . The bia


The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the  S. cerevisiae and humans -UTP was purchased from Moravek Biochemicals. All chromatography materials are from GE Healthcare. Oligonucleotides were ordered from Integrated DNA Technologies (IDT) and synthesized plasmids were ordered from Genewiz. All other chemicals and enzymes were purchased from Fisher Scientific.-UTP. The RNA was purified using Nucleobond Xtra Midi anion exchange gravity columns (Macherey and Nagel). In brief, the column was washed with 100 mM Tris-acetate (pH 6.3), 10 mM MgCl2, 15% ethanol, 300 mM KCl. Following washing, the RNA was eluted in the same buffer with 1150 mM KCl. The eluted RNA was subjected to isopropanol precipitation and was resuspended in water. The concentration and specific activity of the RNA was determined by A260 absorbance (https://www.fechem.uzh.ch/MT/links/ext.html) and scintillation counting, respectively.Radioactive RNAs were created by transcribing in the presence of  is the concentration of the titrated species, S. cerevisiae H/ACA snoRNPs from highly purified components including in vitro transcribed snR30 [In analogy to H/ACA snoRNAs directing pseudouridine formation, snR30 is also hypothesized to form a snoRNP by binding two copies of the core four proteins Cbf5 (dyskerin or Nap57 in humans), Gar1, Nop10, and Nhp2 (L7Ae in Archaea) . Previoued snR30 .D), the binding curve was fit with a hyperbolic function . Thereby, we determined that the full-length snR30 interacts with a subnanomolar affinity with the H/ACA proteins , internal hairpin (snR30ΔIH), or both (snR30 Δ5ʹΔIH). For all three snR30 variants, the affinity of the RNA to the four H/ACA core proteins remains unchanged  and snR30 ΔIH (KD = 12.8 ± 2.4 nM) or the other variants of snR30 (KD = 4.7 ± 0.9 nM) as with snR30 supporting the hypothesis that the binding of Utp23 to snR30 in the absence of other factors may not be specific.Utp23 binds to the snR30 RNP  hairpin , but theof snR30 , Table 3snR30 binds two motifs, rm1 (801–806) and rm2 (836–841), on the 18S rRNA in a sequence-dependent fashion . This reTo characterize the binding of the snR30 RNP to ES6 and the surrounding helices H21 and H22/23 of the 18S rRNA, varying rRNA fragments were constructed. Each ES6 construct includes one or more hairpins of the mature 18S rRNA fold . The radThe longest construct C2-H22/23 spanning nucleotides A661-C962 includes parts of H21 up to the entire H22 and contains both the rm1 and rm2, the C2 site and H22 which could also interact with snR30. Here, the radiolabeled rRNA is titrated against a constant concentration of snR30 full-length RNP. Fitting the data to the hyperbolic function, a low nanomolar dissociation constant was determined  do not form high-affinity interactions with snR30 RNP on their own. Besides this specific, tight binding to rm1/rm2, we also discovered that the snR30 RNP can bind unspecifically and with high stoichiometries to other RNAs such as tRNA, but this interaction is characterized by a comparably low affinity in the high sub-micromolar range.Met. Unexpectedly, Utp23 has an affinity below 2 nM for this tRNA (Utp23 has been reported to bind to the 18S rRNA within ES6H3 and H22 . Therefohis tRNA . Howeverhis tRNA , Table 5During ribosome biogenesis, many RNA and protein assembly factors cooperate and interact with each other such as the snR30 RNP and Utp23. As the snR30 RNP binds rm1-rm2 tightly and specifically, but Utp23 displays tight unspecific RNA binding, we asked whether Utp23 can further increase the affinity of the snR30 full-length complex to the C2-H22/23 rRNA fragment. To test this hypothesis, Utp23 was incubated with the snR30 full-length RNP in an equimolar ratio, and then titrated with C2-H22/23 rRNA in nitrocellulose filtration experiments. Interestingly, we determined a dissociation constant of 20 ± 5 nM and an amplitude of 101 ± 6% for the interaction of the complex of snR30 RNP and Utp23 with this rRNA fragment (in vitro reconstitution of the S. cerevisiae snR30 RNP from highly purified components and the quantitative characterization of crucial RNA-RNA and RNA-protein interactions of snR30. Using this highly defined experimental system allows us to dissect the functional importance of reported snR30 interaction representing a first critical step towards elucidating the molecular mechanism of snR30. Furthermore, we present the quantitative analysis of the ribosome biogenesis factor Utp23 interacting with snR30 and rRNA. Ultimately, this approach allowed us to begin reconstituting critical steps during ribosome formation by assessing the combined effect of the snR30 RNP and Utp23 on rRNA binding. In summary, this work represents a proof-of-concept for gaining mechanistic insight into the molecular interactions during ribosome biogenesis.Here, we report for the first time the in vitro reconstituted snR30 RNP enables future studies further addressing its structure, function, interactions, and molecular mechanism during ribosome formation.We show that the core H/ACA tetrameric proteins Cbf5-Gar1-Nop10-Nhp2 bind to snR30 with a sub-nanomolar affinity , Table 2In addition, we demonstrate that highly purified Utp23 binds in a tight, yet non-specific fashion to RNA , Table 3The high affinity of Utp23 for RNA was also unexpected as previously the affinity of Utp23 to snR30 was estimated to be in the low micromolar range based on electrophoretic mobility shift assays ,21. ThesRegarding interactions with pre-rRNA, the reconstituted snR30 RNP allowed us to probe for the relative importance of interactions of this ribonucleoprotein with different regions of the rRNA during ribosome biogenesis , Table 3Moreover, we determined the affinity of Utp23 to rRNA fragments and again observed an unexpected high affinity, yet non-specific RNA binding. This tight RNA binding by Utp23 was observed both for rRNA constructs containing its reported binding site in ES6H3 and H22 in 18S rRNA as well as other RNAs that did not contain this region , Table 5S. cerevisiae fitness at cold temperatures [In summary, we have characterized important protein-RNA and RNA-RNA interactions between Utp23, snR30 and rRNA that determine the specificity and affinity of recruiting ribosome assembly factors such as Utp23 and the snR30 RNP to the nascent pre-rRNA during ribosome biogenesis. Such quantitative biochemical studies can in the future be expanded to further explore the interactions and functions of the snR30 RNP, Utp23, and other related ribosome biogenesis factors during pre-rRNA folding and processing. In particular, it will be interesting to probe interactions of snR30 and Utp23 with Utp24 which is responsible for pre-rRNA processing at sites A1 and A2 . Similareratures ,30. In cClick here for additional data file.


NEED—The effect of dimensional variability of sheet thickness (tolerance) and tool misalignment is poorly understood for the clinching process. Finite element analysis (FEA) is valuable but requires a lot of and is difficult to verify in this situation due to the asymmetrical geometry and nonlinear plasticity. OBJECTIVE—The objective of this work was to determine the effect of thickness tolerance, tool misalignment and sheet placement (top vs. bottom) in the clinching process, by use of analogue modelling with plasticine. METHOD—Experiments used a scaled-up punch and die, with plasticine as the analogue. Thickness tolerances were represented by sheet thicknesses of 11 and 7 mm, 12 and 8 mm, 8 and 12 mm and 13 and 9 mm for upper and lower sheets, respectively. Two types of lubricant were tested between sheets: glycerine and silicone oil. Angular variability was also introduced. Measured parameters were interlock  and neck thickness. Analogue results for deformation were compared with microscopy of metal clinching. FINDINGS—The results reveal that the multiscale analogue model is an efficient tool for studying the effect of dimensional deviation on a clinch joint. Thickness tolerance showed a critical relationship with interlock, namely a reduction to about half that of the nominal, for both maximum and least material conditions. Increased angular misalignment also reduced the interlock. Compared with glycerine, silicone oil tests showed reduced interlock, possibly the result of a lower coefficient of friction. ORIGINALITY—This work demonstrates the usefulness of analogue modelling for exploring process variability in clinching. The results also show that significant effects for sheet placement are ductility, lubricant (friction), thickness of samples and tool misalignment. The trends of light-weighting, higher performance and increased functionality are some of the drivers for multi-material, hybrid structures and the need for the joining of dissimilar materials. Furthermore, industries are pressured to use environmentally friendly and energy-saving joining methods. Material joining methods such as welding, riveting, clinching, adhesives and screws are widely in use.Clinching is a relatively new high-speed mechanical joining technique suitable for point joining lightweight sheet materials. The only restriction is their plastic properties . MechaniClinching processes include Die-less Clinching, Flat Clinching, Hole Clinching Technology, Clinch–Adhesive Joint, and others. These processes are still being developed; they require much more research to achieve the point where accuracy, quality, and strength of the joints become comparable to the industry standard . CurrentSince clinching can be used in variety of industries, commercial pioneers in this field  have classified their products by tool characteristics. The first and foremost classification is the final shape of the pressed joint. The fundamental distinction between manufacturers using the clinching process is the method to produce joints, which means different die configurations, including expandable and fixed dies . ClincheThere are a number of factors affecting clinch forming, such as interlock between the two sheets, upper and lower sheet thinning, and reduction in the bottom thickness of sheets. Various studies have considered these factors as the main parameters of the process joinability. Inappropriate values of these parameters could eventually lead to one of the following three failure modes in clinched joints: (1) the joint opens because of inappropriate interlocking between the sheets; (2) fracture in the neck area of the upper sheet through low ductility of the material and thickness of sheet materials; (3) a combination of the previous two modes. Lastly, the crack phenomenon in the bottom of the joints is due to the generated tensile stress that can be prevented by eliminating the groove depth i.e., the flat bottom ,15. AddiMany studies have investigated optimization of this process, while neglecting joint quality ,22,23,24The main concern in clinching process is the quality of each single joint or continuous quality control as the clinching process is sensitive to all deviations, particularly in mass production. Varis  suggestsMisalignment can cause tool damage and reduced joint strength. The joints must be formed perpendicular to the metal surfaces. All of the clinching tool sets within the die must be set up to bottom simultaneously to produce consistent button dimensions for all of the joints. The maximum misalignment of a clinching tool was reported to be three degrees . This prFinite element analysis (FEA) is valuable to analyse the clinching problem, but effortful and difficult to verify in this situation due to the asymmetrical geometry and nonlinear plasticity. Examples of FEA clinch models are ,32,33,34Aluminum and other metal sheets in the clinching process require high stress from the punch, and it is also difficult to control the thickness tolerances. However, these factors can be avoided by using plasticine layers. The plasticine layers require less stress from the punch, and it is easy to control the tolerance in an ideal range. Plasticine can be colored in a variety of ways to increase the experiment’s visibility. Plasticine, like metals, exhibits stress–strain behavior at both high strain rates and temperatures. Applications of plasticine in analogue modelling include stamping, rolling, forging, Friction stir welding (FSW) and rock structure ,39,40,41In this study, a plasticine analogue model was used as a cost-effective alternative. The aim was to straightforwardly accommodate variability in geometric tolerances. The tolerances of interest are thickness and misalignment. Therefore, the feasibility of a plasticine analogue model was investigated to ensure a qualitative model could be produced. Following this, the effect of different configurations of sheet thickness tolerances and tool misalignment on the main quality parameters of clinching, such as interlock  explored . This work had several objectives. The first was to evaluate the representativeness of plasticine as an analogue model for steel clinching, and to find a way to produce repeatable results. The second was to determine the effect of thickness tolerance, tool misalignment and sheet placement (top vs. bottom) in the clinching process. A third objective was to provide a qualitative understanding of the plastic flow features. The overall approach (described in more detail below) was to conduct experiments with both a conventional steel tool and steel sheets, and a 10:1 scaled-up punch and die with plasticine. To compare the qualitative evolution of the force profile during clinching, the punch force was measured in both cases, for representative samples. Thickness tolerances were represented by plasticine sheet thicknesses of 11 and 7 mm, 12 and 8 mm, 8 and 12 mm and 13 and 9 mm for upper sheet and lower sheet, respectively. Two types of lubricants were considered between sheets: glycerine and silicone oil. Angular variability was also introduced. Measured parameters were interlock (undercut) and neck thickness. Analogue results for deformation were compared with microscopy of metal clinching.Firstly, an actual round TOX clinching process for sheet-metal was conducted along with a plastic deformation. The main geometric parameters of the joints, such as neck thickness The most important part of this analogue modelling study was to ensure that the plasticine analogue model could reproduce similar results to the real process. Therefore, experimental tests were conducted to compare and establish the plasticine simulation of the clinching process.® 810 Material Testing System). This setup  was used with thicknesses of 1.2 mm and 0.8 mm for upper and lower sheets, respectively. Sheets of the same thickness were cut from one coil to ensure equivalent mechanical properties. The variation of the thickness over the width of a SPCC-coil has been reported to range from 50 μm to 70 μm. Chemical and mechanical properties of SPCC material were reported by Nourani et al. . The ser® PRESSOTECHNIK. Clinched specimens of 6 mm diameter were made by a TOX® clinching die set consisting of a punch (TOX part # 10.180), a round fixed die (TOX part # 10.25) and an elastic blank holder . A total of five optical microscopy specimens were created using the same process parameters. In all cases, the geometric and process parameters resulted in a robust clinch joint.The punch and die are commercially available in the market, thus the shape and dimensions are determined and standardized by TOX®). Specimens were mounted in resin and polished using colloidal silica to 0.02 μm . The main geometric parameters of the joint were measured using an optical microscope . Next, specimens were etched with 2% Nital solution for imaging via optical microscopy [Joints were cross-sectioned with a low-speed saw , ensuring that the distance between the punch and die after clinching was exactly 7 mm. To prevent air entrapment, a hole of 4 mm was drilled in the die. The blank holder was eliminated from this design because the plasticine was too soft and the material would be entirely warped and squished if it was used. Eventually, various techniques were used to assess the accuracy of the die set in the workshop and the geometrical tolerances of the punch and die were found to be less than ±0.05 mm .Figure Multi-layered plasticine slabs were fabricated under required sizes, representing sheets in the clinching process. Different colors of plasticine manufactured by New Clay Products United Kingdom were employed. Manually, each color of plasticine was rolled to a consistent thickness of 1 mm using a rolling pin and an aluminum stencil frame with dimensions of 100 mm × 100 mm × 1 mm (length × width × depth), see A manual press was used to complete the clinching process for a total of 34 samples of plasticine . In the To prevent sample damage, the die-set was placed in a freezer for two hours to ensure that the clinched plasticine was hard enough to be removed from the die-set. Plasticine layers were cut into two sections using stainless steel wire (SS 316L) with a diameter of 0.2 mm.A digital camera (Canon EOS 30D DS126131 8.2 MP with Canon Ultrasonic Zoom Lens) was used to photograph cross-sections. Subsequently, an optical microscope  was used to measure the bottom thickness, interlock and neck thickness accurately. The first step to measure interlock and neck thickness was to find the interface between upper sheet and lower sheet. Samples were then compared with the cross-section of an actual steel clinched joint.Different configurations of the main experiments are summarized in The approach in this survey was experimental rather than theoretical, and involved:Finally, two sets of experiments (S7 and S8) were conducted to determine the effect of angular misalignment on clinched joints. Cold-rolled steel strips were shimmed between the punch and upper die shoe to produce an angular misalignment. Inserting a 1.5 mm shim on one side of the punch results in a 1.5° misalignment. It was not possible to tilt the punch more than 1.5° as the dowel pins did not allow this. Therefore, two steel spacers with thicknesses of 1 mm and 0.5 mm were used to angularly misalign the punch 1° and 1.5°, respectively .It was necessary to fine-tune the plasticine technique and overcome key difficulties with the analogue modelling. Sixteen clinched samples of 12–8 mm (combination of upper sheet 12 mm and lower sheet 8 mm thick) were tested to establish a reliable analogue model. Damage and tears in the plasticine samples during the process were studied to improve the analogue modelling technique and find the appropriate clinching parameters including lubricant, temperature and process time. The preliminary results of the plasticine clinching process, which are of considerable importance to reproduce a damage free plasticine joint, are depicted in A: Damage due to extraction and flash due to cutting—Process time 1 s.The flash occurred after using the cutter to cross section the plasticine samples. The results revealed that, if the specimen was not chilled after clinching, extraction damage occurred on both die side and punch side. The key reason is that plasticine at room temperature is too soft and could easily be damaged during the removal of the punch and die. To address this issue, a series of tests were carried out in which plasticine-clinched samples were frozen at time intervals ranging from 0 to 5 h, with the best outcome obtained with a 2-h freezing period following clinching. Another factor is friction between tools and plasticine slabs, which could be improved by lubricating the slabs.B&C: Damage caused by the use of silicon oil to lubricate the upper and lower sheets—Process time 5 s.Different lubricants such as water, glycerin, and silicon oil were tested, and the findings indicated that, while silicon oil with a lower viscosity effectively decreased friction, the chemical interaction between plasticine and silicon oil could soften the plasticine, resulting in sample tearing and damage. Water as a lubricant was also studied, but the results were inadequate in the initial modelling attempts; therefore, it was excluded from the list. This was consistent with findings from other investigations .D: Damage due to excessive cooling of the sample on die side—Process time 5 s.In other tests, the upper and lower slabs were cooled to −4 °C before the clinching process. These samples were placed in the freezer for 1 to 4 h. Damage was attributed to embrittlement of the plasticine. It was presumed that the damage occurred during the early stages of the clinching process, when the material was in tension. This method could be beneficial to compare the analogue model with the actual clinching process in hard material sheets. However, in this study, the focus was on ambient temperature and ductile materials.E: Damage due to preheating the slabs before clinching—Process time 5 s.Preheating the slabs before clinching was conducted using an oven to evaporate glycerin. The process included heating the oven up to 60 °C in half an hour, putting the plasticine slabs inside the oven for two hours and then cooling down the samples in the room temperature. However, the material became brittle and it was easily damaged.The plasticine clinching process should be performed at room temperature and any freezing or preheating the slab before clinching is not recommended in this application.Using glycerin instead of silicon oil and water as the results were more reliable.The clinching process was timed from 1 to 10 s, and the results indicated that 1 s was surprisingly the best process time to avoid damage.Clinched samples were kept in the freezer for two hours with plastic bag and desiccant which could absorb the moisture in the freezer.After conducting the above tests to search for a reliable analogue model, the improvements below were used to produce consistent results without damage for further experiments:The shear stress between the plasticine layers was one of the key challenges in this investigation. When compared to genuine sheet metal clinched joints, it was essential to ensure that the friction between the layers was sufficient to create a reliable analogue model that accurately simulated an actual sheet metal clinched joint. Additionally, in order to obtain consistent results, the base plasticine model must be calibrated. This was accomplished through the conduct of four sets of experiments and discussion of the results.Results of neck thickness greatest . FurtherFollowing this, a set of experiments (S2) was conducted with two slabs that were cross sectioned in order to compare the results of the neck thickness The main purpose of this set of experiments was to study the sensitivity of the plasticine multi-scaled model to number of layers for each slab in the clinching process. However, using only two slabs of plasticine instead of multiple layers could be faster and easier, especially in studying the thickness tolerance and tool misalignment. Further work is required to establish this.Lubrication refers to the type of lubricant used and its effect on the interlock. Lubricant serves many purposes, but the ultimate goal of a lubricant is to reduce unwanted friction (the resistance encountered when solid surfaces slide against each other). This friction reduction was accomplished by separating two solid surfaces with a thin layer of lubricant. Adding lubricant between the plasticine slabs, punch and die could avoid the damage during extraction of the sample. The coefficient of friction for silicone is believed to be about 0.15  and for Tentatively, this implies that, for clinching in steel, it might be possible that oily surfaces might result in less interlock, hence poorer quality joints. This also raises questions about the role of surface coatings more generally, such as electragol and preprint, as these may be softer in shear than the steel substrates.The focus of this section is to compare the overall geometric equivalence and microstructure evolution of the base plasticine model and actual clinch joint.The cross sections of a 12–8 plasticine joint (base model) were compared to a clinched joint made from SPCC mild steel: sheet thicknesses 1.2 mm and 0.8 mm, identical tool geometry to 10:1 scale, sectioned polished and etched (see method). Observations were made with an optical microscope. The main regions in the cross section of clinched joint were examined: base material, pre-necking region, necked region, stretched corner and basal plate. Results are illustrated in Inspection reveals that the plasticine deformed sample had the same structural anatomy as SPCC mild steel in terms of orientation of colour bands and grains, respectively. The results also had a good agreement with other microstructural studies of steel clinched joints ,47,48. IIn addition, the main quality parameters of the mild steel and plasticine joints were measured. The average values of the neck thickness A significant force is required to complete the clinching process in sheet metals. Elastic deformation and deflection  occurs in the clinching machine structure due to the increased force . As a reThe modified form of the punch force and displacement of the SPCC sheet metal clinched joint was determined per Coppieters et al. , and resEven though the blank holder was not included in the plasticine analogue model, plasticine clinch joints followed a similar pattern to steel clinch joints, see Force is an indication of the rheology, how the sheet material flows into the mould volume provided by the punch and die. This is a complex process because the early stages involve elastic deformation in bending of the sheets , followed by the development of in-plane tensile deformation that culminates in a necking process. Towards the end of the process, there is bulk compression of the material and lateral flow to fill the groove and conform to the cavity created by the tooling. In addition, the circular nature of the geometry adds circumferential strain effects. The strain regime changes throughout the process, as evident in the changes (some abrupt) in the stiffness profiles of the above figures. The rheology represented in the final joint is a consequence of complex interactions between these various temporally changing strain fields.Two sets of experiments (experiments S5 and S6) were conducted to investigate the effect of thickness tolerance in clinched joints by adding and removing the layers on both upper sheet and lower sheet. There are three types of material conditions considered: the nominal material condition is the 12–8 joint sample; the maximum material condition (MMC) is the 13–9 joint sample, and the least material condition (LMC) is the 11–7 joint sample. In The study’s final step involved angularly misaligning the punch. It is encouraging to qualitatively compare the tool misalignment results in this study with that found by Varis  who caseLoss of wall thickness on the advancing side, and gain of wall thickness on the retreating side.Loss of interlock on the advancing side, to the point of total collapse thereof.Severe thinning compression of the upper sheet at the advancing side. This may be due to stretching by the incoming punch. The bottom sheet does not have noticeably different compression.Complete filling of bottom groove at the advancing side and incomplete filling of bottom groove at the retreating side.The notable features of the misaligned case are . Designers should be cautious about racks of clinched joints that might all be misaligned similarly and oriented unfavorably compared to external loads.Even though punch and die were manufactured with an accuracy of 0.05 mm, there are still errors in the punch and die which will affect the results of plasticine.Consideration has not been given to the influence of room temperature and indoor humidity on the plasticine material.There is a thickness tolerance of each group of plasticine sample, and the mean tolerance range is ±0.1 mm.The effect of blank holder was neglected.➢Establishing the FEA model to evaluate the results of plasticine clinched joints according to this study;➢Studying the effect of blank holder force on plasticine analogue model;➢Studying the effect of preheating on plasticine analogue model;➢Investigating the impact of different lubricants in plasticine analogue model;➢Studying the effect of misalignment on tools life using force measurement;➢Utilizing a 3D-printed plastic tool for the plasticine analogue.This study dealt with establishment of a method for plasticine clinching. The feasibility of the plasticine analogue model in reproducing the behavior of clinching process was explored. Furthermore, the influence of sheet-metal thickness deviation and tool misalignment as potential difficulties in the clinching process in production lines was investigated by using the plasticine multiscale model. There are, however, still many technical issues to be explored. Furthermore, the literature review showed that, in each aspect of this process, there are just a few studies which in time can be expanded to better understand press forming. What follows are just some ideas that could be the focus of future studies: With regard to the objectives, this study has confirmed the representativeness of plasticine as an analogue model for steel clinching. In particular, the results are comparable regarding interlock, neck thickness, force profile, and evolution of the strain and its representation in microstructure.The effect of thickness tolerance, tool misalignment and sheet placement (top vs. bottom) has been investigated. The plasticine results show significant effects for sheet placement are lubricant, thickness of samples and tool misalignment. Compared with glycerine, silicone oil has a lower coefficient of friction, which may be the cause of the reduced interlock. Additionally, the optimal stacking of sheets is a 12–8 sample  with 1.87 mm of interlock among four test samples. Moreover, thickness tolerance showed a critical relationship with interlock, namely a reduction to about half that of the nominal, for both maximum and least material conditions. Furthermore, with an increase of the angular misalignment of punch, the interlock decreases. There is confidence that these findings also apply to steel clinched joints because similar effects are reported in the literature ,46 and iThe plasticine also allows a qualitative understanding of the plastic flow features. The evolution of the force to displacement (stiffness) relationships for steel and plasticine show identical qualitative characteristics. This indicates that the rheology of the process is similar in both mediums.This work makes a novel contribution of providing a qualitative appreciation of the effects of sheet placement, sheet thickness tolerance, and tool misalignment on the quality of clinched joints. The results have been demonstrated for plasticine analogue modelling, and comparison with clinched steel sheets shows that the cross sections are qualitatively similar. The study provides some confidence that the principles and relationships described may be applicable to other materials. An advantage of the plasticine method is that it allows the intra-sheet behaviour to be visualised. In contrast, this is difficult to do with metallic materials and the smaller scale of commercial clinching.plasticine analogue model can be calibrated to a range of hard to soft sheet metals by adjusting temperature and lubricants;layers would move on each other if the process time increases as the friction between the layers causes damages at the top layers;controlling the thickness tolerance for values under 0.1 mm is so difficult. therefore, using a multiscale model would be very helpful;friction has a significant effect on the interlock of clinch joints.Plasticine analogue modelling provides a useful method for characterising the deformation involved in a clinch joint. Furthermore, it also reproduces many of the force characteristics: