This paper introduces the Deictic Navigation Network, a cognitive-linguistic framework to analyze and clarify the nature of viewpoint disturbances in language, applied to schizophrenia. We argue that such disturbances have linguistic counterparts in the use of deixis: linguistic elements of which the interpretation relies on the situational context of the discourse and their connection to a subject-bound perspective. The DNN connects such linguistic phenomena to three viewpoint disturbances, which can manifest in different degrees of extremity: (i) the reduced capacity to recognize one’s own subjective perspective and the subjective perspectives of others; (ii) the reduced capacity to separate present perspectives from distinct past, future, and hypothetical perspectives; and (iii) the reduced capacity to integrate projected viewpoint structures into the actual here-and-now. We explain how application of the DNN to language in schizophrenia enables the localization of perspectivization disturbances and helps to clarify the nature of disturbances in the ability to build complex viewpoint structures in language as well as cognition. Schizophrenia is commonly conceptualized as a so-called “self-disorder” , meaningperspective which express subjects’ position in relation to others, the world and themselves  reason about and anticipate their beliefs, intentions, and desires. Empirical studies have demonstrated ToM disturbances in schizophrenia patients, connecting them to various prominent psychotic symptoms like delusions and thought disorder . Each interaction revolves around such a common ground that is built upon the accumulating utterances in the discourse and feeds upon general knowledge, culturally constituted schemata and know-how about the world, language, and social conventions , the Speaker – as primary origo – selects herself-as-secondary-origo-in-the-Narrative-Domain, to which this particular stretch of discourse is to be deictically anchored  (Linde will go to the party), the Speaker represents this subject’s origo in the Narrative Domain, thus construing the event from this subject’s perspective or, depending on lexical choices, of a third subject’s perspective that is either made explicit (Linde will come to John’s party) or remains implicit (Linde will come to the party) (Linde said that John would come to the party), a new Speech Act domain is embedded which gives rise to a new Narrative Domain with new projected and latent origos  . Alternae party) . Note tht origos ; this isdeixis indicates “those aspects of language whose interpretation is relative to the occasion of the utterance: to the time of the utterance, and to times before and after the time of utterance; to the location of the speaker at the time of the utterance; and to the identity of the speaker and the intended audience” (today and next week and demonstratives such as this and that). Note that in generic, expository utterances, this origo is non-subjective by nature; compare for instance the following headline of a Dutch news story: “A woman from Vriezenveen has died because of the fact that she was struck by lightning” , time (past related to an abstract now-point), and causality (objective relation between lightning strike and death).The different origos of Speaker, Hearer, and (possibly) others are all potential vantage points to which the Speaker can anchor linguistic expressions. For an origo to become activated as subjective starting point in language, it must be embedded in deictic structures . The conudience” . More spghtning” , p. 207.deictic anchoring to explain that disturbances in deictic structures in schizophrenic language reveal deviant ways in which patients relate their viewpoint to aspects in the world, including entities, events, locations, and time. Indeed, deixis is not limited to linguistic categories such as pronouns. Rather, it is the functional principle that connects all language to situations  space and time, such as modeled in the DNN. tuations . Since s4Building on the notion of deictic anchoring as introduced by how linguistic expressions are connected to the three distinctive perspectivization requirements, i.e.: how can a study of language in schizophrenia help us understand which requirements are met and which are not and, by implication, help us gain insight into the precise nature of cognitive disturbances in perspective-taking? In the remainder of our article, we will propose that such understanding can be arrived at through application of the DNN. Considering recent work on the phenomenology of schizophrenia , it is pProposing that disturbed deictic navigation is indicative of disturbances in the three perspective-taking requirements introduced by Not acknowledging a bodily viewpoint that is explicitly one’s own and not seeing the other as a similarly viewpointed being reflects a radical form of self-disturbance, in that I-ness and You-ness are not adequately represented and separated, which finds its linguistic counterpart in a hybridity of subjects within the direct environment. This disturbance is located at (i) in we are all heroes here, aren’t we now? (b) If you think that, why don’t you take my place? (c) But then we never fear a place we’ve never been, so you would probably agree to do so, thinking it’s all my fault.5(a) But 6 Therefore, a lack of epistemic expressions that moderate the attribution of feelings or perceptions to either Speaker or Hearer, and the objective, instead of subjective, construal of causal connections could also point at Speech Act Domain disturbances  while at the same time presuming that “you,” in a conditional state , would not, and in a generic condition  would indeed take her place. The mixed references make it unclear whether I and You are (to be) represented as separate subjects with each their own origo and corresponding subjective orientation, or not. In addition, refraining from acknowledging one’s own and other’s (bodily) perspectives equals the negation of the concept of perspective as a whole.urbances . Consideurbances .Not being able to project origos in the Narrative domain and separate those origos from origos in the speech act domain reflects another radical disturbance. In this case, one is not able to separate I-ness here-and-now from I-ness-in-the-past/future/virtuality. This is represented by route (ii) in Love, and how important it is for everybody to love everybody”), came the delusion of all delusions, the main thought that would continue to dominate my thinking for years to come. (b) The thought was that of a young man, the person whom I had last been involved with in a romantic way. (c) The sentence immediately made me think of my friend, who for some strange reason became the cause of all causes! (d) He knew all the answers, I thought! (e) This was not just some answers to some things, but all the answers about everything to do with life! (f) How could someone be so dumb, you say, to come to such a conclusion? (g) I don’t know that. (h) All I can say is that I have always been considered an intelligent person, with talents and abilities that have been noted by others. (i) It was not my intelligence, but rather my sense of religion that might be compared to one persuaded by a cult. (j) But in this case, my mind came to this conclusion all by itself. (k) Sometimes it makes me feel that I just can’t handle all of the complexities of life. (l) But up to that point I had been excited about life; there were so many new challenges in my life, and I had just come to a big city and had fallen in love with all it had to offer.7(a) After the sentence extraordinaire , where “came” projects the origo in the Narrative Domain, and (k) which is a generic utterance which is anchored by “makes” and “can’t” in the Speech Act Domain, again alternated with the Narrative Domain in (l) which is signaled by the deixis of “that point.”In this excerpt, the Speaker uses present perfect , past (i–j), present , and past perfect  tense interchangeably to indicate how she felt at the onset of her illness. In doing to, she displays skill in the complex interplay of tenses that indicate different, and at times contradictory and confusing viewpoints on the time-line within the Narrative Domain. Yet, the boundary crossing between Speech Act and Narrative domain, as to where a particular stretch is (to be) represented (or in both at the same time), is not always clear. In (i), the Speaker’s origo is anchored in the Narrative Domain by “Being unable to integrate projected viewpoints from the Narrative Domain in the actual here-and-now indicates a third radical disturbance. This action is represented in him, for already he has “cured” my hallucinations, but my depression prevents me. (d) You can’t be as smart as the next man if you haven’t had like experiences.8(a) I am a dependent on science. (b) My sanity depends on what fumbly old men can come up with in the area of discovery. (c) I could praise In this excerpt, it is difficult for the Hearer to anchor the subject depicted by the Speaker in the Narrative Domain. This subject is referred to in (c) by the underspecified pronouns “him” and “he,” which makes it unclear whose origo(s) are (to be) represented in the Narrative Domain as the subject who “has cured my hallucinations” (c), since the subsequent sentence (d) does not resolve this either. As a result, the projection of the origo of “he”  in the Narrative Domain is hampered through the use of an underspecified pronoun, which in turn inhibits the integration of this projected viewpoint into the present in which the Speaker could praise “him” for his past deed.9 Such outcomes would advance our understanding of the nature of perspective taking disturbances in schizophrenia.In developing and applying the Deictic Navigation Network (DNN) to language in schizophrenia, we aim to enable the identification of localizable perspectivization disturbances, which is of importance as it could reveal and clarify fundamental disturbances in one’s ability to build complex viewpoint structures in language as well as cognition . DNN-anaUltimately, results would shed a light on gradual differences in viewpoint taking abilities, and do justice to the rich and complicated ways in which humans navigate (linguistic) intersubjective relations. An advantage of this approach is that the DNN accounts for various inherently different types of viewpoint disturbances, which can manifest in different combinations, thus allowing for in-depth insights into the nature of perspectivization issues at the level of individuals, and of specific patient groups.Note that each of the disturbances will amount to a fourth, radical disturbance: an incapability to represent situations from both a global and a participant viewpoint and negotiate between the two . When inLvS, KvK, and JS provided substantial contributions to the conception and/or design of the work and the drafting and revising of the work. They provided approval for publication of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Homologous domains embedded in multidomain proteins of different domain architectures (DA) may exhibit subtle, but important, differences in their structure and function. Here, we consider two multidomain proteins, Arf nucleotide binding site opener (ARNO) and G protein‐coupled receptor kinase 2 (GRK2), which have very different DAs, but both contain pleckstrin homology (PH) domains. We analyzed the roles of residues selectively conserved in these subfamilies of PH domains from ARNO and GRK2 proteins. DA‐specific residues in PH domain are found to contribute to structural and functional specialization of ARNO and GRK2 in terms of (a) specific intra‐ and interprotein interactions; (b) specificity for phospholipids; and (c) participation in conformational excursions, leading to various functional forms. Our approach can also be applied to subfamilies of other protein families to identify subfamily‐specific residues and their specialized roles. Arf6ADP‐ribosylation factor 6ARNOArf nucleotide binding site openerDAdomain architectureGRK2G protein‐coupled receptor kinase 2PHpleckstrin homology domainHomologous proteins are known to perform same or similar functions, but their functional levels, specificity to ligands, and regulatory mechanisms may vary among homologues Pleckstrin homology domain is named after the protein pleckstrin, which is found in platelets Pleckstrin homology domain is very versatile in nature and is known to be tethered with many protein domains Arf nucleotide binding site opener protein consists of two structural domains, a Sec7 domain followed by a PH domain as shown in Fig. ARNO protein exists in three structural forms Auto‐inhibited state is maintained by a self‐regulatory mechanism, wherein the major differences between them are the conformations and hinge dynamics There are two isoforms of ARNO that have been crystallized and structure determined in full‐length form GRK2 protein has three structural domains, namely RGS, protein kinase, and PH domain as shown in Fig. In this work, we have classified the PH domain sequences into two subfamilies based on their occurrence in ARNO and GRK2 proteins, which are characterized by different DAs. We recognize residues in PH domain that are selectively conserved in one subfamily compared to the other and explore their structural and functional roles.The two datasets used in the analysis are sequences of homologous ARNO proteins and sequences of homologous GRK2 proteins with their respective DAs conserved within ARNO and within GRK2 homologues.Pfam database http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit) and are removed from the list of sequences for analysis to result in nonredundant datasets corresponding to two different DAs. We safely used a 90% sequence identity cutoff as some of the identical proteins show < 100% sequence identity due to additional few residues in one compared to the other either in N‐terminal end or C‐terminal end or both.A script was needed that uses the DAs of ARNO and GRK2 proteins as a query and searches in the Pfam database to identify homologous proteins with same DA. This script should be such that it seeks to avoid entries with incomplete/partial domains and long insertions between domains that may correspond to a domain that is yet to be recognized and documented in the domain databases. Therefore, we developed our own script to recognize PH domain containing sequences corresponding to two different DAs with the other desired features mentioned above. The Perl scripts developed are provided in Appendix The dataset has been further refined by removing proteins with overlapping domains, incomplete domains, and domains with long insertions.jalviewThe PH domain region of homologous sequences of ARNO and GRK2 was multiply aligned as depicted in Fig. naccess1u29 and 1u27 and visualized using pymolThe conserved residue columns or conservatively substituted residue columns between the ARNO homologs and GRK2 homologs in the master alignment (CSA3) were identified, and its corresponding residue columns in SA1 and SA2 with more than or equal to 85% conservation have been considered as DA‐specific residue columns. A conserved residue column in SA1, but with a semi‐conservative or % residue conservation > 50 in its corresponding residue column with same residue or residue type in SA2 and vice versa was not considered to be DA‐specific residues. Intraprotein and protein–protein interactions were identified, from the Protein Data Bank entries 2r09, 4kax of ARNO and 5ukl of GRK2 proteins and using Protein Interactions Calculator The final dataset consists of 88 homologous sequences of ARNO and 21 homologous sequences of GRK2.The conserved residue columns or conservatively substituted residue columns in the PH domain region of ARNO homologs and GRK2 homologs with the cutoffs described in the Methods have been considered as DA‐ or subfamily‐specific Fig. . HencefoFurther, solvent‐buried residues of PH domain of ARNO and GRK2 were identified as described in Methods and the corresponding aligned residue columns were not considered for further analysis, as buried residues are generally known to be situated in the core maintaining the tertiary structural integrity of the domain.There are 16 of the alignment positions in the PH domain that are conserved or conservatively substituted across the members of ARNO and GRK2. As these are conserved across all the PH domain sequences from ARNO and GRK2 subfamilies, these are not DA‐specific residues. Out of these, 10 of the residues are also buried and hence could be contributing to the structural integrity of the domain fold. Two other residues, Trp‐281 and Trp‐285, maintain the conformation of the inositol binding pocket. Similarly, residues Lys‐273 and Gly‐275 interact with phosphoinositide and its equivalents are also part of the binding site region for phosphoinositide If a residue in an alignment position is conserved only within ARNO members or only within GRK2 members in the datasets, then such residues are considered to be DA‐specific.Using this criterion, 40 DA‐specific residues were identified for ARNO PH domains and 21 DA‐specific residues for GRK2 PH domains. If in an alignment position different residue types are conserved within ARNO (say Ser) and within GRK2 , these residues also qualify as DA‐specific residues for ARNO and GRK2, respectively.The structural and functional roles of DA‐specific residues for both ARNO and GRK2 are provided in Tables As mentioned before, the ARNO protein exists as three different conformers and Arf6 protein can interact with ARNO protein only to its partially active form, whereas it is inhibited in inactive form. This switch between inactive and partially active forms is maintained mainly by PH domain and its proximal regions in the protein Six DA‐specific residues  are involved in domain–domain interactions in the inactive form of ARNO, whereas two DA‐specific residues (Asp‐266 and Ile‐379) are involved in partially active form of ARNO Table ; Fig. S2One of the major features in determining the ARNO functional forms is the C‐terminal helix. The C‐terminal helix undergoes change in its orientation between the functional forms of ARNO. In inactive form, this helix interacts closely with PH domain and maintains a steric hindrance for Arf6 interaction for its interaction with PH domain. In the partially active form of ARNO, this C‐terminal helix undergoes a change in its orientation, thereby permitting Arf6 to interact with PH domain The conformation and interactions of some of the residues play a role in the helix orientation as shown in Fig. The PH domain of ARNO binds to the cell membrane by mediating interaction with phospholipid head and helps to recruit other proteins to the membrane. The splice variant 2G of ARNO protein is known to interact with Ins  P4 There are nine DA‐specific residues involved in interaction with Ins  P4 in 2G variant and seven DA‐specific residues from 3G variant to interact with Ins  P3. Although both phosphoinositides bind in the same binding pocket, with six DA‐specific residues common between them, it does not imply that the DA‐specific residues are playing same roles. The two phosphoinositides bind in slightly different orientations, resulting in differences in interaction pattern. Despite being the part of same binding pocket, the DA‐specific residues interact with the phosphoinositides in different ways, explaining their vital role in providing specificity toward phosphoinositides.Figure The partially active form of ARNO in its auto‐inhibited state allows Arf6 to interact with activator site. Upon accessing the activator site in PH domain and its proximal elements, it relieves the auto‐inhibited state of ARNO through allosteric activation. ARNO, upon interaction with Arf6, gets activated and turns from auto‐inhibited state to fully active state Arf6 interaction with PH domain seems to bring allostery‐mediated conformational change in ARNO. As mentioned already, re‐orientation of C‐terminal helix is noticed in the transition between inactive and partially active forms of ARNO. C‐terminal helix undergoes further change in orientation between partially active form (auto‐inhibited state) and active form of ARNO as shown by superposition of the two conformers of ARNO Fig. A. Along Loss of main chain–main chain hydrogen bond between Asp‐382 and Ser‐378 and main chain–side chain hydrogen bond between Asn‐291 and Ile‐379 seem to be related to re‐orientation of the helix  P2 and PtdIns  P2 are known to bind GRK2 PH domain. It is known that GRK2 PH domain has two binding pockets for phosphoinositide, where the loop1–loop3 forms one and the loop1–loop5 forms the other Pleckstrin homology domains are known to have a highly conserved fold and a general property of phospholipid binding and localization to the membrane by associating with βγ subunits of heterotrimeric G proteins. In the current work, two of the subfamilies of homologous PH domains were defined on the basis of their occurrence in two different DAs in two multidomain proteins, ARNO and GRK2. Though there are several residues that are conserved completely across these two subfamilies of PH domain, DA‐specific residues were identified in the two sets of PH domain sequences. We identified potential reasons for the DA‐specific conservation. We note that many of these residues in ARNO and GRK2 PH domains are involved in intraprotein interactions with domains and domain–domain linkers outside PH domain. This observation is consistent with the fact that ARNO and GRK2 have very different DAs. Further, some of the DA‐specific residues are found to form variable intraprotein interactions facilitating structural excursions among inactive, active, and partially active forms of ARNO. Some of the DA residues are also identified to confer specificity to specific phospholipids.Our work provides a convenient general framework to understand the residue determinants of subfamily specific functional and structural features for subfamilies in other protein domain families too.The authors declare no conflict of interest.NN performed all the analyses of sequences and structures and wrote computer programs. RS and NS conceived the idea and designed the project. NN wrote the first draft of the manuscript. All the three authors agreed on the final version of the manuscript.Fig. S1. Multiple SA of PH domain regions of ARNO and GRK2 sequences.Click here for additional data file.Fig. S2. Two different conformers of ARNO in auto‐inhibited state.Click here for additional data file.Fig. S3. Phosphoinositide binding with ARNO PH domain.Click here for additional data file.Fig. S4. ARNO interaction with Arf6.Click here for additional data file.Fig. S5. Residues that confer phosphoinositide interaction in PH domain of GRK2.Click here for additional data file.Appendix S1. Perl scripts to search the Pfam sequence database for multi‐domain proteins of specific domain architecture.Click here for additional data file.


Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid–liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases. FUS gene was initially identified as a fusion oncogene on chromosome 16 in human liposarcoma [Fused in sarcoma (FUS) is a DNA/RNA binding protein containing 526 amino acids. The osarcoma . The mososarcoma ,5,6. As osarcoma ,8. The pFUS is well-studied for its transcriptional regulation function in cells. FUS binds to single-stranded DNA motifs in the promoter region of certain genes, which are transcribed by RNA polymerases II and III, and then accumulates near the transcription start site (TSS), resulting in changes of transcription levels ,10. AddiFUS modulates RNA splicing in conjunction with splicing regulators or precursor mRNAs . FUS is During DNA damage repair, FUS is one of the proteins firstly recruited to the DNA damage site ,23. LossIn 1999, Baechtold demonstrated that FUS participates in DDR, which promotes complementary ssDNA annealing and single-stranded oligonucleotide uptake of homologous supercoiled DNA to form a D-loop. The D-loop formation is a principal step in DNA double-strand break repair via recombination, and the oncogenic fusion form FUS–CHOP does not promote DNA pairing . Wild-tyFUS associated with ALS was discovered in 2009 and later on FUS-immunoreactive cytoplasmic inclusions were found in ALS–FUS patients [FUS mutations have also been detected in patients with essential tremor (ET) and Parkinson’s disease. For example, a mutation in its exon (c.868C > T) leads to a stop mutation during FUS expression (p.Q290X), resulting in a potential cause of ET [Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) is a well-known neurodegenerative disease caused by the loss of both upper and lower motor neurons, first described by the French neurologist Jean-Martin Charcot in 1869 . The firpatients . Shortlypatients ,32,33. Fse of ET ,35. Intese of ET ,37. Furtse of ET ,39,40,41se of ET .FUS has two possible models for cytoplasmic aggregation leading to neurological diseases. One is the gain-of-function model, in which FUS gains a toxic function in the cytoplasm. FUS may sequester important regulators or trigger abnormal signaling pathways to alter cell physiology . The altFUS belongs to the FET/TET family, in which proteins share domains similar in both structure and function . FUS conThe binding of FUS to RNA may be related to the ZnF motif. The RGG–ZnF–RGG domain is thought to be the major RNA-binding sequence ,51. FurtPrions are self-replicating proteins with misfolded conformations and can lead to diseases, also known as infectious proteins. A single prion can act as a template to fold soluble proteins containing the same amino acid sequence into the prion conformation, resulting in self-replication . Prions The experimental results from the interactions between FUS and polyglutamine inclusions and the predictions suggested that the FUS prion-like domains are located in the N-terminal QGSY domain and are part of the first RGG domain (amino acids 1–239), with an additional region in the RGG3 domain (amino acids 391–405) ,67. By uPhase separation is a physical process in which a supersaturated solution of components spontaneously separates into two phases with different densities, both stable and coexisting . This phStudies have shown that FUS has three forms of existence—dispersion, droplets, and hydrogels—and the reversible dynamic phase transition is driven by the low-complexity (LC) domain at the N-terminus of FUS . AccordiThe basis of the reversible phase separation of FUS is the self-assembly of the LC domain . Once thRecent solid-state nuclear magnetic resonance (NMR) studies have indicated a structurally ordered amyloid core in the LC domain of FUS, also known as the prion-like domain. At the same time, it was discovered that the residues 39–95 constitute the structurally ordered core of the prion-like domain fibers in combination with the computational assistance method based on the MCASSIGN algorithm ,90. The The overall bone architecture of residues 43–95 and the orientation of the side chains of residues 44–52 and 65–95 have been determined, while residues 55–62 form a dynamic loop. The fragments of the β-strands forming the cross-β contain residues 44–46, 52–54, 62–64, 67–70, 85–90, and 93–95, determined from the characteristic arrangement of the backbone carbonyl and the fiber growth axis .Liu et al. found that 37SYSGYS42 and 54SYSSYG59 in the amyloid core region 39–95 can form reversible fibers that are sensitive to temperature and phosphorylation, termed the reversible amyloid nucleus . ReversiThe physiological function of FUS is performed while in a droplet state. In vitro, FUS droplets are amorphous, and it is speculated that, in vivo, FUS may have no ordered structure, and the oligomers formed may be gradually decomposed ,100. CurNumerous studies have shown that the regions of FUS other than the LC domains, especially the C-terminal domain, are important in the phase transition behavior of FUS. Qamar et al. demonstrated that the synergistic cation–π interaction between the tyrosine of the LC domain and arginine of the structured C-terminal is related to phase separation . The eleThe nuclear import receptor TNPO1 and Kapb2 regulate FUS phase separation by binding to FUS via its C-terminal domain . They exFUS are associated with ALS, accounting for about 5% of familial cases (FALS) and 0.7–1.8% of sporadic cases (SALS) [FUS mainly occur in exons 3–6 and exons 12–15. According to statistics, approximately two-thirds of FUS mutations are in exons 12–15, which encode ZnF motif, RGG2 and RGG3, and nuclear localization signaling domains. In particular, more than 50% of ALS-associated FUS mutations were found in the C-terminal NLS domain. Exons 3–6 encode the QGSY-rich and RGG1 regions, and about one-third of the mutations are in them [FUS are shown in It is currently known that over 50 mutations/deletions in s (SALS) . Mutatio in them ,88,109. FUS mutation enhances the interaction between FUS and SMN and reduces its levels at the normal expression sites, thereby impairing motor neurons [Unlike the mechanism by which the mutated FUS locates in the cytoplasm, the FALS-associated  neurons . Primari neurons . Mutatio neurons . The stu neurons ,149. In  neurons . This veMost neurodegenerative diseases are associated with the misfolding of proteins. A growing number of studies have shown that FUS mutations in prion-like domains mediate the aggregation of FUS, which is an important reason for the development and progression of most neurodegenerative diseases. The mechanism of action of prion-associated regions of FUS needs to be further investigated, such as under what conditions the pathological aggregation of FUS occurs and whether they can play a role in disease progression by allowing the cell–cell metastasis of pathological protein aggregates. Although emergency particle assembly and prion proliferation has been confirmed, whether FUS mutations associated with neurodegenerative diseases cause normal function loss, a gain of toxic properties of FUS aggregates, or a combination of the both requires clarification and further studies. In addition, based on known mechanisms, the research of small-molecule-targeting inhibitors of FUS is also a topic of particular interest regarding finding a cure for related diseases. We are optimistic that with the deepening of FUS research, this field will make a major breakthrough.


The paper presents the results of investigations in which high-density polyethylene (PE-HD) was maintained in deformation by means of a special grip (holder). The wear of the sample was significantly higher than that of the undeformed sample. This effect persisted even after partial relaxation of the stress in the sample after 24 h. Additional investigations were carried out to explain the obtained results. There were the microscopic observations of the surface after friction, measurements of microhardness, and surface free energy. Changes in the value of surface free energy and a significant decrease in microhardness with deformation under tension were observed. Deformed materials have a different surface appearance after friction and a different size and form of wear products. It was indicated that it is probable that the cohesion of the material will decrease and that the character of the wear process will change as a result of tension. Deformation under tension without heating of polymeric material (PE-HD), e.g., as a result of assembly, has been qualified as a threat to be taken into account when designing and analysing polymeric sliding elements.Polymer materials are increasingly being used for sliding machine elements due to their numerous advantages. They are used even where they are deformed and in such a state that they interact frictionally, e.g., in machine hydraulics or lip seals. Few publications deal with the influence of deformation, which is the effect of, e.g., assembly on tribological properties of polymeric material. This deformation can reach up to ε  Polymer-based sliding elements are increasingly being used in mechanical engineering. This is a result of their favourable properties such as a possibility to work without lubrication, low coefficient of friction when working with steel, high chemical resistance, easy shaping, and low cost of producing elements, especially in mass production. Polymer sliding elements also appear where they are deformed during tribological interaction. Stryczek et al. conducted tests on the use of polymer materials in machine hydraulics . The resIn the literature, there are many publications describing attempts to obtain a polymeric material with more favourable tribological properties by tension. This type of treatment consisted in the introduction of significant deformation values amounting to several hundred percent. They were carried out by drawing polymeric plates at elevated temperature ,5,6 duriFew works concern the relatively low value of the deformation obtained at room temperature, i.e., the situation corresponding to the deformation resulting from the assembly. Cayer-Barrioz et al. studied the interaction of a steel roll with a strained polymeric rope . The polThe authors decided to investigate the influence of cold deformation on the tribological properties of polymeric materials for four main reasons: firstly, increasing the number of polymer-sliding elements appearing where they are deformed during tribological interaction. Secondly, there is few information in the literature about the influence of cold deformation on the tribological properties of polymeric materials. Thirdly, the phenomena and processes occurring during the cold deformation of polymeric materials can influence the friction and wear of the polymeric material . FinallyThe works carried out included tests of wear, microhardness, and surface free energy. The results of tribological tests showed that the wear of PE-HD significantly increases after the polymer material is deformed by tension. The studies on microhardness and surface free energy were designed to provide information that would help to explain the behaviour of the polymer. In order to obtain information on the influence of deformation on the adhesive component, tests of surface free energy were carried out. Microhardness measurements helped to evaluate the influence of deformation on the cohesion of the polymeric material.® G AST natural  was used. Preliminary investigations have shown that this polymeric material is susceptible to deformation under tension and that its wear after applying tension significantly increases. This polymer is used for the production of sliding elements such as bushings and seals.In the studies, high-density polyethylene PE-HD (PE 300) PolystoneThe samples for tribological tests during tensile testing had a shape similar to the outline of the so-called “paddle” presented in the EN ISO 527:2012 standard for the determination of tensile properties of plastics. As the space at the test stands was limited, the shape of the sample was reduced and slightly modified. The research was carried out during frictional interaction in the direction parallel and perpendicular to the direction of the tensile force vector. Therefore, two types of samples suitable for both situations were prepared . SamplesWhen a polymer sample is strained at room temperature, e.g., on a tensile testing machine, and then removed from the grip and left freely, the deformation may change. This is due to the viscoelastic properties of the polymeric materials. Depending on the type of polymer and the strain value, the sample can be fully or partially restored to its original form. Sliding elements and seals remain deformed after assembly. To reproduce this situation, the polymer samples ought to remain in a deformed state during the test; therefore, they had to be held in grips to prevent them from returning to their original form.Special handles with PLA (polylactide) callipers were designed and manufactured in three-dimensional printing technology . The calThe changes in callipers spacing and, thus, in the tension of the sample were achieved by turning the nuts on the bolt. The nuts in the tension grip were turned manually. Therefore, it is difficult to precisely determine the deformation rate of the samples. Based on the time during which the deformation took place (turning the nuts) and the obtained value of the deformation, it can be estimated that the deformation took place at a speed of about 5–10 mm/min.The samples used in the study had the shape of so-called “paddles”. The part between the grip and the measuring part was rounded. Due to variable stiffness along the length of the sample in areas adjacent to the gripping parts, the strain after tension was different from that at the centre of the measuring part. For this reason, deformation was not determined by the distance between the callipers but was checked at the place where it was intended to be measured during tribological tests.ε. This way of determining the strain allowed us to obtain a precise value of the strain in the place where further measurements were made, i.e., microhardness, surface free energy, or wear.The value of deformation was determined by measuring the distance between the imprints of the indenter used to measure microhardness using the Knoop method. The imprints were oriented in a direction perpendicular to the direction of the tensile force vector. The initial distance between markers  was deteThe tests were carried out to determine the deformations that could occur during assembly. Four values of tensile strain were adopted: 2%, 5%, 20%, and 50%. For deformations below ε = 2%, in the polymers, elastic deformations prevail. According to the recommendations, the deformation of plastic parts in machines and equipment should not exceed 2%. Therefore, a deformation range of up to 2% can be defined as acceptable, tolerable, and safe. A 5% strain is outside the acceptable range. Such deformation may occur, among other things, in an improperly mounted element. Tensile strain of 20% can occur during assembly in some machine parts, e.g., in the lip seal of the shaft. In the terms of 50% strain, it is difficult to find a machine element in which such deformation would be the result of the assembly. However, this deformation may be achieved by deliberate deformation of the sliding element. Tests conducted for 50% strain can provide information on whether cold deformation significantly affects the tribological properties of the analysed polymer and whether it can be used as a technology used in the production of sliding elements. In addition, measurements taken for such a high deformation may contribute to a deeper explanation of the friction and wear of deformed machine elements.Wear tests were carried out on a test stand in the combination of a roll and a block. The rotating steel roll worked with a polymer sample kept in a deformed state . The volCt = 0.25; Lm = 1.25). The speed was 0.33 m/s.The step motor shaft was equipped with a 30-mm diameter and 3-mm wide stainless steel X8CrNiTiS18-9 roller. The roughness of the cylindrical surface of the roll was Ra 0.85 ÷ 1.25 µm , the average pressure was about 3 MPa. The load on the roll was 49 N. The friction path was selected experimentally so that the length of the groove during the tests does not exceed the assumed value of 5 mm. This length of groove ensured a sufficient distance between the roll and the edge of the sample. The friction path was 2000 m.Kw is often used by manufacturers of sliding materials and given in the product documentation, it was decided to present the wear results using this ratio. The Kw indicator is determined by the following formula:V is the volume of worn material (mm3), FN is the normal force (load) during friction (N), and S is the friction path (m).As the wear coefficient The results of the wear tests show that the direction in which frictional interaction was carried out was important for the deformed polymer. Therefore, a method that would provide information depending on the setting of the indenter was sought for hardness testing. The position of an indenter relative to the material is important in the Knoop method because the indenter is a diamond-shaped pyramid . When meShimadzu HMV-2 microhardness tester was used during the research. The measurements were carried out by maintaining the samples in a deformed state using a grip . For theThe measurements included tests in the parallel and perpendicular directions to the tensile force direction. Due to the fact that the results of microhardness tests turned out to be very significant, the tests for more strain values  were conducted.In order to evaluate adhesion in the combination of deformed polymer–steel, tests of surface free energy were carried out. The measurements were taken directly after applying tension to achieve 20% and 50% strain and after 30 min of maintaining the deformation ε = 50%. The Kruss DSAHT12 goniometer was used in the study. Drops of four liquids were applied on the polymer sample: distilled water, diiodomethane, ethylene glycol, and formamide. The angle between the droplet outline and the surface of the polymer sample was measured using a grip to maintain the sample deformed. The value of surface free energy was determined using the Owens–Wendt–Rabel–Kaelble (OWRK) method.Surfaces of interacting elements are subject to different changes resulting from processes and phenomena occurring on the contact surface during friction. The condition of the surface subjected to friction can give a lot of information helpful in explaining them; therefore, it was decided to include microscopic observations in the research. The investigations of sliding surfaces of polymeric samples after interaction with a steel roll were carried out with the use of a scanning electron microscope (SEM).The space in which the sample was placed was very limited. The grips described above were too large to fit into the microscope socket. The polymer was kept stretched during microscopic observations owing to a special base produced using a 3-D printer a.The sample was stretched in a standard grip b. Two hoA drawback was observed in the method described above. Namely, after the procedure of transferring the sample to a smaller grip, the deformation was reduced by “removing the clearance” between the screw and the inner wall of the hole in the polymer. Measurements showed that, for initial sample deformation ε = 20% after cutting off the gripping parts, the final deformation was about 17–18%. Despite the decrease in strain value, microscopic observations showed differences in surface appearance after the process of friction between deformed and undeformed materials. The assessment of the surface condition after friction was carried out for an undeformed polymer and after stretching to 20% strain.x) and the expanded uncertainty (U):U of 95%), S is the standard deviation, and n is the number of measurements. The results of the research were given in a form x ± U.In terms of tests for each variant , the measurements were carried out for four polymeric samples. Immediately after the deformation, the sample together with the grip was mounted on the test stand. If the grip was placed on the stand incorrectly , the result of the measurement was significantly different from the others. The Dixon test (Q test) was used to eliminate such results (vitiated with a gross error). Other results were used to calculate the mean value  increases as the deformation increases.The results showed that the deformation of polymeric material has a significant influence on its wear. The wear coefficient . Maintaining the deformation caused an increase in the value of free surface energy by about 10%, which in the end constituted about 16% more than for the undeformed sample .ε = 20%, the size of wear products was much greater as compared to undeformed polymer .Changes in polymer properties occur already at deformation resulting from tension up to 2% strain. In this situation, microhardness decreased by 17% and wear increased by 51%. This is important information as the deformation range up to 2% strain is considered safe and acceptable when designing various elements.The direction of interaction in relation to the tension force vector is important. Greater changes concern the sliding interaction in the parallel direction. The tensile wear of PE-HD was significantly higher when applied parallelly to the direction of the tensile force than when applied perpendicularly.Maintaining PE-HD in a deformed state with applied tension for a certain period of time results in a different material behaviour than in measurements carried out immediately after deformation. In the terms of PE-HD, after a time, the wear was higher than in the case of measurements carried out directly after deformation. This is important, for instance, for seals that are deformed during assembly and function as they are during their lifetime. The described effect does not diminish with time; the seal can be characterized by increased wear even after a considerable period of time after assembly.In terms of polymer properties, the deformation is unfavourable. It causes a significant increase in wear and a decrease in microhardness. The deformation of a polymer material does not appear to be a way to improve its properties. It ought to be treated as a threat that must be taken into account when designing sliding elements that are strained during assembly.After deformation due to tension, the changes include both friction components describing the metal-polymer interaction, i.e., the mechanical and adhesive component. Any attempt to explain the effect of tension on friction and wear ought to refer to both of these components.


Crotalus adamanteus venom, named PDE_Ca. (2) Methods. The PDE_Ca structure model was produced and validated using various software . (3) Results. The proposed model of the enzyme indicates that the 3D structure of PDE_Ca comprises four domains, a somatomedin B domain, a somatomedin B-like domain, an ectonucleotide pyrophosphatase domain, and a DNA/RNA non-specific domain. Sequence and structural analyses suggest that differences in length and composition among homologous snake venom sequences may account for their differences in substrate specificity. Other properties that may influence substrate specificity are the average volume and depth of the active site cavity. (4) Conclusion. Sequence comparisons indicate that SVPDEs exhibit high sequence identity but comparatively low identity with mammalian and bacterial PDEs.(1) Background. Snake venom phosphodiesterases (SVPDEs) are among the least studied venom enzymes. In envenomation, they display various pathological effects, including induction of hypotension, inhibition of platelet aggregation, edema, and paralysis. Until now, there have been no 3D structural studies of these enzymes, thereby preventing structure–function analysis. To enable such investigations, the present work describes the model-based structural and functional characterization of a phosphodiesterase from  Proteucleases ,6,7,8,9.ucleases ,10,11,12ucleases ,14. Phos+, NADP+, and GDP [Phosphodiesterases (E.C. No. 3.1.4.1) belong to the Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family of metalloenzymes . General and GDP . Because and GDP ,18.PDEs have been isolated from various snake venoms, including Deinagkistrodon acutus , BothropSnake venom PDE was first reported by Uzawa . This isSVPDEs are monomeric proteins of high molecular weight 98–140 kDa), with basic pIs (8.4–9.2), and metal cofactors, usually zinc, which are essential for catalytic activity ,36,37,38 kDa, witCrotalus adamanteus venom. The PDE_Ca structure model was produced and validated using various software . The sequence alignment, structure-based substrate specificity, maturation, and comparison with PDEs from other organisms are also discussed. Although the amino acid sequences of PDEs from various snake species are available in the literature, there is no information regarding their three-dimensional (3D) structures. Therefore, it is difficult to correlate function with structure. In order to enable structure-function studies, here we present a model-based 3D structural characterization of the phosphodiesterase from Ca precursor contains 851 amino acids with 830 amino acid residues in the mature form. Sequence alignment indicates high sequence identity among SVPDEs and comparatively low sequence identity (<65%) with their mammalian counterparts , Zn+2 2 , and Ca+2  (PDE_Ca precursor numbering scheme) are fully conserved among all phosphodiesterases examined, except N751 and H755 in SVPDEs, where these have mutated to D751 and R755, respectively, in mammalian homologs , somatomedin B-like domain (residues 81–124), ectonucleotide pyrophosphatase/phosphodiesterase domain  (residues 147–479), and the DNA/RNA non-specific domain (residues 534–867) (The primary structure of mature PDE_534–867) . The regCa is fully conserved, with other proteins in the Protein Data Bank (PDB) containing a similar structure fold  online web server  for domaure fold . The molectively . The ther SVPDEs ,45,46,47Ca contains 60 asparagine (N) residues , of whiotparam, . The prion sites . PDEs ofectively ,49.The homology model for three-dimensional (3D) structure characterization was produced using various online modeling servers  [Ca has a complex structure. It is a multi-domain protein that consists of four domains—a somatomedin B domain, a somatomedin B-like domain, an ectonucleotide pyrophosphatase/phosphodiesterase domain , and a DNA/RNA non-specific domain (The 3D structure of PDE_rfamily) ,61,62,63c domain . These dThe somatomedin B domain (SMB) is located at the N-terminus of the protein and comprises amino acid residues 33–79 . It has The somatomedin B domain connects to another domain called the Somatomedin B-like domain (SMB-like). This domain consists of residues 81 to 124 . Like thBoth the SMB and SMB-like domains are highly compact. Disulfide bridges are arranged in the centers of both domains, forming covalently bonded cores .The SMB-like domain connects to the catalytic domain, called the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP/PDE) domain, through a short connecting segment of 22 amino acid residues (125–146) .+21) is coordinated by four amino acids , and the other one (Zn+22) is coordinated by three residues . All these catalytic amino acid residues are fully conserved in mouse NPP1 [The ENPP/PDE domain consists of amino acid residues 147–479 . It contuse NPP1 , human Nuse NPP1 , and bacuse NPP1   is coordinated by amino acid residues Asp153, Thr191, His359, and Asp358 (+22) is coordinated by amino acid residues Asp311, His315, and His476 , ENPP2 (Human Autotaxin), Xac Nucleotide Pyrophosphatase/Phosphodiesterase (Xanthomonas axonopodis), and Taiwan cobra (Naja atra atra) PDE (PDB ID: 5GZ4 and 5GZ5) shows that the amino acid residues coordinating these metal ions are fully conserved  and Bothrops atrox (PDE_Ba) were modeled, using the same modeling and validation programs used for the PDE_Ca structural model. The atomic coordinate of human Ectonucleotide pyrophosphatase/phosphodiesterase 3  was used as a template. The Ramachandran plot analysis indicates that in both the modeled structures, 98% of amino acid residues were in the favored region of the plot, and 2% were in the allowed region , and Bothrops atrox (PDE_Ba) were compared, taking into account the active site residue composition, active site cavity volume and average depth, and the surface charge distribution  to access the active site of PDE_Ca, while preventing it for PDE_Vl.The average active site cavity volumes of PDE_ectively . These vCa  is highly positive   show a high preference for ATP.Another factor that affects the substrate specificity of these enzymes is the surface charge distribution . The surpositive A, for PDnegative B. Analysrastes )  show a hCa, human ENPP3 [Vipera lebetina (PDE_Vl), Bothrops atrox (PDE_Ba), and the PDE from Taiwan cobra  shows that the three-dimensional structural folds of these enzymes are similar and that all of them align well, with an RMSD value range between 0.21 and 0.92     . They haof 0.61)  and disuof 0.61) . Howeverof 0.61) . For thiof 0.61) A–G, whicof 0.61) C–F. The of 0.61) .Ca has a close evolutionary relationship with SVPDEs and PDEs from human beings and mice  [Ca contains 851 amino acid residues [PDE_zymogen) ,45. The residues    [http://www.ncbi.nlm.nih.gov/protein). The signal peptide was identified using the SignalP 3.0 server [Ca was used as a query for searching homologous proteins from the non-redundant database by searching with the NCBI Protein BLAST using default parameters. The multiple sequence alignment of the selected homologous protein sequences, including the amino acid sequence of PD_Ca, was generated using MUSCLE [The amino acid sequence of PDE_0F7Z2Q3)  was obta0 server  with defg MUSCLE . The alig MUSCLE .The Weblogo 3.2 ,78 was uCa primary sequence was analyzed for the presence of domains/motifs using the conserved domain search tool [http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. The protparam and Compute pI/MW tools from the ExPASy Proteomics server (http://web.expasy.org/compute_pi/) [I) and molecular weight of the protein.The PDE_rch tool , availabute_pi/)  were useCa, while putative glycosylation sites were predicted using the NetNGlyc 1.0 Server [The 3DLigandSite-Ligand binding site prediction Server  was used0 Server  and Scan0 Server , with deCa.The DiANNA web server  and DinoCa was generated using various online proteins modeling programs, such as I-TESSER [The 3D model of PDE_I-TESSER , the MODI-TESSER , and SWII-TESSER , using tI-TESSER . The finI-TESSER .Ca was validated through MD simulation using various programs, like AMBER16 [++ [2. The system was then heated gradually from 0 to 300 K for 250 ps with a constant atom number, volume, and temperature (NVT) ensemble, at the same time that the protein was restrained with a constant force of 10 kcal/mol. Å2. The equilibration step was carried out using the constant atom number, pressure, and temperature (NPT) ensemble for 500 ps, and the simulation was done for 100 ns with a 4 fs time step. The temperature and pressure were kept constant at 300 K and 1 atm, respectively, by Langevin coupling. The long-range electrostatic interactions were computed with the Particle–Mesh Ewald method (PME) [The modeled structure of PDE_ AMBER16 , GROMACS AMBER16 , MDweb,  AMBER16 . The all AMBER16 . The webER16 [++  was usedod (PME) , keepingCa was validated using the PROCHECK software [The build model of PDE_software ,54, ERRAsoftware , and Versoftware ,57.Ca protein model was aligned to homologous proteins using the PyMOL molecular graphics visualization program [The build PDE_ program .Charge and radius calculations were carried out using the PDB2PQR server program . The surCa was carried out using various computational biology programs. The amino acid sequence comparison analysis indicated that SVPDEs display high sequence identity (90.6%) with one another and comparatively low sequence identity (58.33%) with mammalian and bacterial PDEs. The three-dimensional model of PDE_Ca, produced using various modeling programs, was of good quality, as shown by the PROCHECK and ERRAT analysis. The modeled structure was further analyzed by molecular dynamic simulation, and the analysis indicated that all important structural parameters, such as chirality, disulfide bonds, and the absence of steric clashes, were correct. The root mean square deviation and radius of the gyration did not suffer significantly during model building and were maintained at 1 Å and 31.7 Å, respectively. The structural analysis indicated that the complex structure of PDE_Ca is folded into a multi-domain protein that comprises four domains—a somatomedin B domain, a somatomedin B-like domain, an Ectonucleotide pyrophosphatase/Phosphodiesterase domain , and a DNA/RNA non-specific domain. Structural comparisons with PDEs from other snake venoms, and mammalian and bacterial counterparts indicated that the surface charge distribution and the average active site cavity volume and depth vary considerably, which may contribute to their variable substrate specificity. Finally, during the maturation process, venom PDEs lose their signal and activation peptides to convert into the fully active mature forms. The structure of PDE_Cα presented in this paper is only a predicted structure. These conclusions need to be confirmed with experimental evidence [In conclusion, a sequence and structural analysis of PDE_evidence .


Its photocatalytic activity for the degradation of methylene blue (MB) was determined. The results suggest that the Cu7S4 microcages with nanotwinned building blocks possess higher catalytic activity than the Cu7S4 microcages without the nanotwinned structures, suggesting that the special nanotwinned components can improve the catalytic performance of the multipod framework. Further investigate reveals that the nanotwins inside the Cu7S4 microcages can facilite the transport of free charges, decrease the recombination of photoinduced electrons and holes, and elongate the lifetime of the electron–hole pairs. Our work will provide powerful evidence that the nanotwinned building blocks of the synthesized Cu7S4 microcages play a crucial role for the high catalytic activity.The 14-pods Cu Chowdhury et al.  crystals as sacrificial templates. However, the structure-controlled synthesis of different building blocks in Cu7S4 hollow nanocages with polycrystalline shells is rarely studied. In the present work, we have developed an ethanol assisted sacrificial Cu2O template approach to the shape-controlled synthesis of nanotwinned Cu7S4 microcages with Multipod Framework shell by an ethanol assisted sacrificial Cu2O template approach. The structure-dependent photocatalytic performances and mechanism of the hierarchical Cu7S4 hollow microcage assemblies with twinned nanoplate building blocks are determined and discussed in detail.Copper sulfide, a non-stoichiometric 2 2H2O , EDTANa2 ·2H2O , NaOH , Na2S·2H2O  n-butyl alcohol , and ethanol . All the reagents and solvents for synthesis were purchased from commercial sources and used as received without further purification.The chemicals used in this study included Cu (Ac)2O template was synthesized as described in the previous report . Their morphologies and structures were imaged by scanning electron microscopy  and transmission electron microscopy , respectively. The valence states were determined by X-ray photoelectron spectroscopy . The UV–vis absorbance spectra of as-synthesized samples were collected on a Hitachi UH-4150 (Japan) spectrophotometer, using BaSO4 as reference. The UV-vis absorption spectra of MB solution were recorded on a Model 2501 PC spectrometer . The electrochemical impedance spectra (EIS) were carried out in a traditional three electrode system. The prepared sample was used as working electrode, a platinum plate (99.9%) was used as the counter electrode, a saturated KCl Ag/AgCl electrode was used as the reference electrode, and 0.5 M Na2SO4 solution as the electrolyte. The photoluminescence (PL) spectra were studied on Hitachi F-7000 fluorescence spectrophotometer equipped with an excitation wavelength of 370 nm.The phases of Cu2O2  was added into 4 mg/L methylene blue (MB) solution (250 mL), and then no catalyst, 12.5 mg 14-pod Cu7S4 hollow particles without the nanotwinned structures and 14-pod Cu7S4 hollow particles with the nanotwinned structures were dispersed into the solution, respectively. The final mixture was stirred for 30 min in a dark box to reach the MB adsorption equilibrium. A 2 mL MB solution aliquot was sampled every 15 min and measured for ultraviolet-visible (UV-vis) absorption spectrum.To determine the photocatalytic activity of the obtained hollow particles, 2.5 mL H7S4 hollow particles. All the diffraction peaks are indexed to the standard monoclinic structure of Cu7S4. No peaks of impurities, such as copper oxides or other copper sulfides, are detected, suggesting the high purity products have been obtained.7S4 hollow particles exhibit two strong XPS peaks at 932.8 and 952.16 eV, corresponding to the orbits of Cu 2p3/2 and Cu 2p1/2, respectively of Cu+  and interiors (bright), which confirms the formation of hollow structures  of the HRTEM image reveals serious image tailing of the reflection fringes –(4) (Sun et al., Based on these results, the possible photocatalytic mechanisms of the 14-pods Cu7S4 hollow particles can facilitate the transport of free electrons and improve the lifetime of electrons and holes. The 14-pods Cu7S4 hollow particles with nanotwinned building blocks exhibit a smaller capacitive arc radius and weaker peak intensity of PL than 14-pods Cu7S4 hollow particles without nanotwinned building blocks. Therefore, the nanotwinned structures greatly contribute to the high photocatalytic activity.Huang et al. , Chen et7S4 hollow photocatalyst with nanotwinned structures that possess superior photocatalytic activity for the degradation of MB dye. The exceptional photocatalytic activities are comprehensively attributed to the nanotwins inside the Cu7S4 hollowcages that can promote the transport of free charges, inhibit the recombination of photoinduced electrons and holes, and extend the lifetime of the electron–hole pairs. The 14-pods Cu7S4 hollow particles with nanotwinned building blocks exhibits a smaller capacitive arc radius and weaker peak intensity of PL, indicative of the promoted charge transfer, which further evidences the above conclusion. Our work is of great importance in the synthesis of high-active hollow photocatalyst with nanotwinned structures. These unique 14-pods Cu7S4 hollow cages can not only enrich the family of copper sulfide architectures, but also offers a good opportunity to understand the fundamental significance of nanotwinned structures. It is expected that hollowcages with nanotwinned building blocks can exhibit outstanding properties in various fields.In summary, we have successfully synthesized the multipod frameworks of CuAll datasets generated for this study are included in the article/XL planned and supervised the project. HZ, YX, and PC performed the experiments. HZ and PC wrote the first draft. ZZ and WM revised the manuscript. XL deeply revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recombinant human P-stalk greatly stimulates GCN2. Both domain II of uL10 and the C-terminal tails of P1 and P2 are necessary for maximal GCN2 activation. On actively translating ribosomes, the C-terminal tails of P1 and P2 are sequestered by elongation factors, suggesting P-stalk availability could link translational stress to GCN2 activation.General control nonderepressible 2 (GCN2) phosphorylates eIF2α, regulating translation in response to nutritional stress. Here, we show that although tRNA stimulates purified, recombinant human GCN2 in vitro, mammalian ribosomes are even more potent GCN2 activators. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) showed GCN2 interacting with domain II of the uL10 P-stalk protein. The P-stalk is a uL10/P1 Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR. A dynamic balance of anabolic and catabolic cellular pathways interlace to efficiently use available resources. The integrated stress response (ISR) enables cells to recognize a variety of imbalances in key cellular resources and respond by activating protein kinases that modify the cell’s translational and transcriptional programs. The ISR is widely conserved across eukaryotes and responds to numerous stressors including amino acid insufficiency, protein misfolding, high salinity, UV light, and glucose starvation . GeneralMet, forms a ternary complex that delivers the initiator tRNA to the small ribosomal subunit to form the 43S preinitiation complex (The eIF2 (αβγ) heterotrimer, together with GTP and the initiator methionyl-tRNA complex . Transla complex –9.m2 ppGpp . Under am2 . This muin cells , 18. Howin cells , 19. Forin cells , 21. GCNin cells , 22. Mor P-stalk  and Sind P-stalk .While GCN2 can be activated in cells, a wide range of observations suggest that the enzyme is maintained in an inactive state in the absence of stimulation , 17. Yea26ArgUCU) and the putative ribosome recycling factor GTPBP2  , 34. Sho33Here, we have reconstituted activation of human GCN2 in vitro using purified components. We show that human GCN2 interacts directly with ribosomes and by using a combination of hydrogen/deuterium exchange–mass spectrometry (HDX-MS) and truncation analysis, we have identified domain II of the ribosomal P-stalk protein uL10 [previously known as P0 ] as the Sf9 cells and purified it to homogeneity  and assayed GCN2’s ability to phosphorylate it in vitro using a phospho-Ser51–specific antibody . Human GCN2 binds to deacylated tRNA with a KD of ∼2 μM , which is similar to yeast GCN2 . This tRNA interaction was abrogated by two mutations in the previously characterized tRNA-binding m2 motif in the HisRS-like domain of GCN2 , implying tRNA is binding to the m2 motif as previously shown in yeast . Deacylated tRNA did not further stimulate GCN2 beyond that seen with ribosomes alone. Thus, ribosomes show ∼20-fold greater potency and ∼3-fold higher maximal stimulation than observed with deacylated tRNA.In marked contrast to 10 µM tRNA, 50 nM purified ribosomes dramatically increased the rate and extent of eIF2α phosphorylation C. A titrGiven the remarkable activation of GCN2 by ribosomes in the purified, reconstituted system, we attempted to characterize the interaction between GCN2 and ribosomes. Recombinant, full-length, StrepII-tagged GCN2 was combined with rabbit reticulocyte lysate and fractionated by size using sucrose gradient sedimentation. A subset of GCN2 comigrated with the ribosomes in the cell lysate D. In a pSI Appendix, Fig. S2; full details in SI Appendix, Table S1) identified the domains of GCN2 that influence ribosome binding. Each recombinant protein was purified, immobilized on StrepTactin resin, and used in pulldown experiments with purified ribosomes. This analysis showed that three GCN2 domains are needed for maximal ribosome binding: the pseudokinase domain, the HisRS-like domain, and the CTD . Comparing constructs D and L shows that the pseudokinase domain contributes to ribosome binding . To detect even weak activation by the various GCN2 constructs, these single-point assays were carried out at 50 nM ribosomes, twice the EC50 for ribosomes. Full-length GCN2 (construct A) was able to phosphorylate eIF2α at a low level in the absence of ribosomes and was significantly stimulated by the addition of ribosomes. The eIF2α phosphorylation was due to the purified human GCN2, since the D858N point mutant that eliminates GCN2 kinase activity still bound ribosomes but eliminated eIF2α phosphorylation . The kinase domain alone (construct B) has similar basal level of eIF2α phosphorylation, but it was not stimulated by ribosomes. Inclusion of the HisRS-like domain (construct K) reinstated ribosomal activation. Other constructs lacking the HisRS-like domain (constructs I and J) phosphorylated eIF2α at a basal level but showed no increase in activity in the presence of ribosomes. The pseudokinase domain of GCN2 seems to modestly contribute to maximal ribosomal stimulation . For constructs K and L, which had greatly impaired binding to ribosomes but had only partial decrease in activity in single-point assays, we performed titrations with ribosomes that confirmed that these constructs are significantly less active than the wild-type enzyme .Using the in vitro eIF2α phosphorylation assay, we examined the ability of each construct that contains the kinase domain to phosphorylate eIF2α both in the presence and absence of ribosomes , indicating that the CTD has a key role in maintaining GCN2 in an inactive state. The charged linker that connects the N-terminal RWD domain and the pseudokinase domain has a minor role in GCN2 repression, as when this is deleted (comparing construct F with E), GCN2’s basal activity increases twofold . These findings demonstrate that in human GCN2 the HisRS-like domain is critical for both ribosome binding and kinase activation. The CTD serves an autoinhibitory function to minimize basal activity, consistent with the autoinhibitory role of the CTD in yeast (Two GCN2 regions appear to have a role in the autoinhibition of the kinase activity in the absence of ribosomes. The first is the GCN2 CTD. Upon deletion of the CTD (construct C), GCN2’s basal activity increased ∼20-fold  at 32 °C . Despite the complexity, the results unambiguously identified a single continuous region of reduced isotopic exchange in the large ribosomal subunit protein uL10 . This protein is part of the P-stalk, a heteropentameric complex that protrudes from the ribosome and forms part of the GAC where the translation factors eEF1A and eEF2 bind. Several peptides of uL10 showed a significantly reduced rate of deuterium uptake in the presence of excess GCN2 . While HDX-MS does not directly map protein–protein interactions, areas showing decreased isotopic exchange rates in the presence of a binding partner are likely to represent the binding site between the two proteins. The peptides identified as containing the interaction site with GCN2 are within residues 121–157 of uL10. This is part of domain II, a unique insertion in the N-terminal domain (NTD) of uL10 that is not present in bacteria. The insertion is on the surface of the ribosome, and it extends from the P-stalk toward the A site, where it can contact translational GTPases [e.g., eEF2 as shown in This is the largest system yet to be examined with HDX-MS, covering 76 proteins digested into 1,070 peptides, representing 65% of the constituent sequences of the ribosomal proteins . P-stalk complexes were coexpressed in insect cells and purified under nondenaturing conditions. Pulldown assays showed that this P-stalk complex is able to bind to GCN2 immobilized on StrepTactin resin (Dataset S2). Consistent with HDX-MS results with intact ribosomes, domain II peptides (121–137 and 145–153) show significant decrease in isotopic exchange upon interaction with GCN2. The isolated P-stalk additionally showed GCN2-dependent decreases in HDX in domain I of the NTD of uL10 that also contacts the 28S rRNA in the intact ribosome . These helices form part of the dimer interface that is necessary for GCN2-mediated translation control . There is also an increase in isotopic exchange in a peptide (residues 630–639) in helix αC of the N-lobe of the kinase domain. This helix is one of the elements that is involved in the switch between the inactive and active forms of the yeast GCN2 , a region near the C terminus of the domain  and in the insert that is not present in the yeast GCN2 kinase domain crystal structure (peptides in the region 662–794) . We also tested whether the P-stalk complex and tRNA had a synergistic effect on GCN2 by including a low concentration (100 nM) of the P-stalk and titrating in tRNA, but we saw no significant difference with respect to tRNA alone , suggesting that there is no synergy. Among the three GCN2 activators we have tested, ribosomes are the most potent, followed by the isolated P-stalk, and then by deacylated tRNA . The influence of the P-stalk on GCN2 is primarily an increase in specific activity, with no change in Km .Beyond a physical interaction, we found that the isolated P-stalk activates GCN2-mediated phosphorylation of eIF2α in the absence of the remainder of the ribosome D. TitratSI Appendix, Fig. S4). In contrast to the full-length P-stalk, the uL10 NTD did not activate GCN2 . Strikingly, ΔC14all P-stalk was incapable of activating for GCN2 . The extent of activation seems to be proportional to the number of CTTs available for interaction with GCN2, with activities in the order 5 CTT (WT) > 3 CTT (ΔC14P1) > 1 CTT (ΔC14P1/P2) > 0 CTT   B.The role of GCN2 in the ISR pathway as a unique convergence point of nutrient availability and cell growth and proliferation has stirred interest in this kinase as a possible target for pharmaceutical development. Much of our current understanding of GCN2 has been derived primarily from in vivo studies in yeast. While aspects of this pathway may be well conserved in eukaryotes, there seem to be key differences in the way that GCN2 is activated in yeast and mammals. Furthermore, the interplay between GCN2 and protein translation means that it can be difficult to disentangle direct versus indirect mechanisms of control using in vivo analysis. For these reasons, we sought to develop an in vitro system to study the mechanistic basis of GCN2 regulation.KD. We see no synergy in activation by P-stalk and deacylated tRNA, which may imply that these two activators operate either in parallel, in different cellular compartments or under different circumstances in vivo.Consistent with a wide range of studies in cells, we find that deacylated tRNA activates human GCN2 toward its substrate eIF2α in vitro. In addition, we find that human GCN2 is even more potently stimulated by ribosomes and the purified ribosomal P-stalk complex. The degree of activation by ribosomes and P-stalk is higher than with deacylated tRNA, which does not achieve maximal stimulation of GCN2 even at a concentration five times its The in vivo activity of GCN2 has been previously linked to ribosomes in both yeast and mammalian systems , 20. Howm2 mutant in the HisRS-like domain cannot be activated by ribosomes in vitro. The homology of this domain to a tRNA synthetase, together with the inactivating m2 mutant, was a major line of evidence implicating deacylated tRNA as a direct activator , as described in full in SI Appendix, SI Materials and Methods.eIF2α phosphorylation was detected by Western blotting with an anti–phospho-eIF2α antibody as described in SI Appendix, SI Materials and Methods.StrepII-tagged GCN2 constructs were bound to StrepTactin resin and ribosomal binding was detected by capturing purified ribosomes. Association with ribosomes was also shown by comigration of GCN2 with ribosomes in a sucrose gradient. For assay of P-stalk components binding to GCN2, purified GST-tagged P-stalk constructs were captured onto StrepTactin-bound GCN2. Details are described in SI Appendix, SI Materials and Methods. Briefly, to detect GCN2 interactions with ribosomes, two reactions were prepared: one with 0.5 μM ribosomes alone and the other having 0.5 μM ribosomes and 2.5 μM GCN2. Both reactions were incubated on ice for 15 min, and then buffer with D2O was added to bring the D2O concentration to 78%, and samples were further incubated for 5, 50, and 500 min at 32 °C. Exchange was quenched, and samples were frozen in liquid nitrogen. Samples to analyze P-stalk complex/GCN2 interactions were similarly prepared, except deuteration was carried out for 0.3, 3, 30, 300, and 3,000 s. Samples were prepared for GCN2 alone, P-stalk complex alone, and two samples of GCN2 plus P-stalk: one sample contained 5 μM GCN2 and 15 μM P-stalk, while the other sample contained 15 μM GCN2 combined with 5 μM P-stalk.Experiments were set up and analyzed as described fully in Supplementary FileSupplementary FileSupplementary File


Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the  Drosophila melanogaster homologue, MSPS (mini spindles) are members of the XMAP215 protein family , also known as CKAP5, and its E. coli cells during recombinant expression. Moreover, there were similar problems of degradation with the TACC domain of the drosophila homologue of TACC3, dTACC. As the chTOG/MSPS CTD is implicated in interactions with the TACC domain it was therefore decided to characterize the CTD of chTOG instead. The importance of the very C-terminal residues in chTOG for binding to TACC3 was demonstrated by the loss of interaction upon deletion of residues 1932–1957 CACB, HN(CO)CA, H(CCCO)NH, (H)C(CCO)NH and HNCO experiments recorded at 700, 800 and 950 MHz on Bruker Avance spectrometers at 25 °C. Side chain resonance assignment was initialized using the backbone-sidechain TOCSY experiments H(CCCO)NH and (H)C(CCO)NH and completed using a combination of HCCH TOCSY and 13C NOESY-HSQC experiments. Spectra were processed with Topspin 3.1 (Bruker) and all assignments were performed with CCPN analysis 2.4 . It was possible to find assignments for all of these even though backbone amide peaks were missing for three of them . Out of a total of 141 backbone nitrogens, 141 α carbons, 131 β carbons, 126 γ carbons, 86 δ carbons, 26 ε carbons and 141 backbone carbonyls a total of 132 (94%), 141 (100%), 131 (100%), 102 (81%), 65 (76%), 25 (96%) and 132 (94%), respectively, could be assigned. 405 out of a total of 423 backbone resonances (96%) and 543 out of a total of 647 sidechain proton resonances (84%) were assigned. In terms of sidechain nitrogen containing groups, all six asparagine δ NH


Data were considered significant at p<0.05. All mandibles that were examined had bilateral MF while unilateral AMF was found in two mandibles (6%). The foramens were mostly oval in shape and their most common position was between the second premolar and first molar and the most common orientation was posterior-superior. The comparative analysis of mandibular anthropometrics showed significant variations (p<0.05) with different ethnic groups. The findings emphasize the ethnic variations and edify that the foramen position is not always as stated in reference textbooks. The clinical creditability of the study is cautioning the surgeons on possible variations of the MF and AMF anthropometrics compared to existing literature in order to avoid any unforeseen injury related to anaesthesia or dental surgeries. Further studies with large sample sizes representing whole country are recommended to establish the standard MF and AMF anthropometrics of Zambian population.The mental foramen (MF) and accessory mental foramen (AMF) are the strategically important landmarks during surgical interventions and anaesthetic nerve blocks procedures involving the mental nerve. The study aimed at evaluating anthropometrics of MF and AMF in Zambian adult human mandibles and it was cleared for ethics from TDRC Ethics Review Committee . A total of 33 Zambian adult human mandibles were evaluated for shape, position, and direction of opening of foramen. All measurements were performed using a Digital Vernier Calliper and statistically analysed for per cent frequency and mean and standard deviations, and we performed the one sample The mental foramen (MF) is an oval or circular opening on the body of the mandible where the mandibular canal terminates. It is an exit for the mental nerve and blood vessels, which are terminal branches of inferior alveolar nerve, artery, and vein. The mental nerve provides innervations of the lower teeth, lip, gingival, and lower face , 2. The nd premolar and 1st molar. The MF was asymmetrically located between the right and left sides and predominantly oval. The direction of opening was mostly superior and posterosuperior and rarely labial, mesial, or posterior [nd premolar on the right side and between 2nd premolar and 1st molar on the left side [nd premolar tooth and bilaterally symmetrical in a majority of cases. Its vertical position was slightly below the midpoint of the distance between the lower border of the mandible and the alveolar margin [The MF is usually located in the body of the mandible at an equal distance from the superior and inferior border below or between the apex of the first and second premolar , 4, 5. Teft side . In a str margin .Any foramen in addition to MF in the body of mandible is known as accessory mental foramen (AMF) and it tends to exist in the apical area of the first molar and posterior or inferior area of the mental foramen. As AMF is due to branching of mental nerve before passing through MF, its shape, size, and verification of its existence would prevent accessory nerve injury during periapical surgery , 10. TheHence location, size, shape, position, and incidence of MF and AMF would facilitate the dental surgeon to apply nerve block in different surgical procedures involving lower jaw. The anatomical research reports on variations in anthropometrics of MF and AMF between race and geographical location signifying the need to establish local values. Currently there are no established values on MF and AMF for Zambian population. Therefore, this study is aimed at evaluating anthropometrics of MF and AMF in Zambian adult human mandibles to establish specific MF and AMF anthropometrics of Zambian population in order to make a gateway to add them to the medical literature.A total of 33 Zambian adult cadavers out of 35 availed in the Anatomy Laboratory of Michael Chilufya Sata School of Medicine (MCS SoM), which met the inclusion criteria, that is, dentate adult mandibles, were considered for the study. The cadavers used in the study were legally permitted for the use of education and research purposed at the MCS SoM and the appropriate permission was obtained from the Dean, MCS SoM for their use in the study.Ethical clearance for this anatomical study  was sought from TDRC Ethics Review Committee , Ndola, Zambia. Handling of cadavers was done only in Anatomy laboratory dissecting hall and no body parts were moved out of the dissecting hall. The cadaver was treated with respect as to the respect given to human life. The information about the cadaver in terms of names or names of the relatives was not sought or collected and only information about age, sex, and race was used for the study.As a minimum, all mandibular teeth from right first molar to left first molar were presentAll mandibular teeth from right first molar to left first molar were in a reasonably normal position and alignmentThe mandibles were disarticulated, cleaned, and then rinsed in 70% ethanol solution and allowed to dry for 24hrs. The mandibles were chosen according to the following criteria:The following observations and measurements were performed directly on each dry mandible for both left and right sides. The measurements were made using Digital Vernier Calliper, which was calibrated before every measurement.Numbers of MF and AMF on each dry mandible for both left and right sides were visually scored and recorded.The greatest horizontal and vertical dimensions of each MF and AMF were recorded.The direction of opening of each mental canal through the lateral surface of the mandible was visually determined and recorded.  Position 1: situated anterior to the first premolar  Position 2: in line with the first premolar  Position 3: between the first and second premolar  Position 4: in line with second premolar  Position 5: between the second premolar and first molar  Position 6: in line with the first molarThe positions of the MF and AMF in relation to the mandibular teeth of the lower jaw were recorded as follows :  PositiThe positions of MF and AMF were evaluated by measuring the distance of MF/AMF (in mm) from various landmarks including symphysis menti, alveolar crest, posterior border of the ramus of mandible, and lower border of mandible .  AB: distance from alveolar crest to lower border of mandible  AC: distance from alveolar crest to upper margin of MF/AMF  BD: distance from lower border of mandible to lower margin of MF/AMF  WX: distance from symphysis menti to posterior border of ramus of mandible  WY: distance from symphysis menti to medial margin of MF/AMF (anterior chord)  XZ: distance from posterior border of ramus of mandible to lateral margin of MF/AMF (posterior chord)  VD: vertical diameter of the MF/AMF = AB – (AC+BD)  HD: horizontal diameter of the MF/AMF = WX – (XZ+WY)The distance measurements included the following: The relative position of MF/AMF was found by subtracting the average distance from symphysis menti to medial margin of MF/AMF (WY) with length of the mandibles, i.e., ratio of WY/WX.The positions of MF and AMF in relation to the mandibular teeth and mandibular anthropometrics from defined positions were compared with reported values of different ethnic groups on different geographic locations, viz., Malawians , Turkish t-test. Data were considered statistically significant at p<0.05.Statistical analysis was done using IBM-SPSS software version 20. The most prevalent shape, position, and orientations were found by simple frequency analysis. The MF and AMF diameters for size and distances from defined positions were measured using a Digital Vernier Calliper (in mm) and statistically analysed for per cent frequency and mean and standard deviations (SD). The comparative analyses of mandibular anthropometrics from defined positions were performed using a one-sampleA total of 33 Zambian adult human mandibles of known sex that included 31 male mandibles and 2 female mandibles were evaluated for MF and AMF anthropometrics measurements. Since the proportion of female to male samples was too low (6%), the data were not analysed and stratified by sex. The frequency distribution of MF and AMF and their shapes in Zambian adult human mandibles are shown in The dimensions of the MF and AMF  on the mThe study found that the most common position  for MF oThe positions of MF in relation to mandibular teeth among different ethnic groups on different geographic locations were compared using per cent frequency  and it wThe frequency distributions of MF and AMF directions of opening (orientation) are shown in The mandibular anthropometric measurements (the distance of MF and AMF from defined landmarks) on Zambian adult human mandibles (n=33) are presented in  t-test comparative analysis  and the rest were presented with bilateral MF. There was no case with absence of foramen and there were no differences in the prevalence of MF and AMF between left and right sides of mandibles. Though our findings are in accord with most of the earlier reports , 12, 23,3 and the et al. , the AMF et al. , Zimbabw et al. , and Mal et al.  adult po et al. , 18 reveThe study also found that the overall mean dimension of MF was 3.35mm, while it was 3.18mm and 3.79mm in the Zimbabwean , 24, 25.The position of the MF varies considerably by geographic location and nationality , 17, 19.Previous studies on mandibles of different ethnic groups , 16, 26 According to the anthropometric measurements in the study, the MF was found to be at an equal distance on both sides of the mandible but it was not true in case of AMF. The mean anterior chord (WY) and posterior chord (XZ) obtained were 28.5mm and 73.4mm, respectively, for MF on both sides of mandibles, while they were 32.6mm and 71.3mm on the left side and 27.9mm and 75.5mm on the right side for AMF. The mean length of mandibles was 101.9mm. It was also noted that the mean position of MF was found 13.86mm above the lower border of the mandible and 15.66mm below the alveolar ridge on both sides of mandibles but the AMF was found 10.9mm and 13.5mm above the lower border of the mandible and 22.5mm and 16.1mm below the alveolar ridge, respectively, on the left and right sides. The relative position of the MF and AMF in the mandible was obtained by calculating the ratio of WY/WX and it was 0.28 for MF on both sides of the mandible but it was 0.31 on the left side and 0.27 on the right side for AMF. Previous studies  reportedA comparative study on mandibular anthropometrics of selected reports of different ethnic groups , 20, 21 As a preliminary attempt, the study has established specific MF and AMF anthropometrics for adult humans in Copperbelt province of Zambia. There are variations in the MF characteristics of studied Zambian mandibles compared to established values worldwide and within the region. The results of the present study highlight the racial differences in the most common position and relative position of the MF observed among different populations in the different geographic locations. The clinical creditability of the study is cautioning the surgeons on possible variations of the MF and AMF anthropometrics compared to existing literatures in order to avoid any unforeseen injury related to anaesthesia or dental surgeries. Although the study has the limitations that the findings are based on a small sample size, which is also having too low female to male proportion which was due to challenges in acquiring cadavers, the data was normally distributed and showed normal variations so the information on anthropometrics of MF and AMF presented in this article can help anatomists, prosthodontists, orthodontists, surgeons, forensic odontologists, and paleoanthropologists to predict the position of the MF and AMF and perform both diagnostics and safer clinical procedures led on the mandibles of Zambian population. However, it is recommended for further studies with large sample sizes representing whole country to establish the standard MF and AMF anthropometrics of Zambians.