In a self-paced reading experiment, the regions before the relativizer  showed reading patterns consistent with expectation-based incremental processing: standard SRs, with the highest constructional frequency and the least complex syntactic structure, were processed faster than the other two variants. However, in the regions after the relativizer and the head noun where the existence of a relative clause is unambiguously indicated, a top-down global effect of thematic ordering was observed: passive SRs, whose thematic role order conforms to the canonical thematic order of Chinese, were read faster than both the standard SRs and the disposal SRs. Taken together, these results suggest that two expectation-based processing factors are involved in the comprehension of Chinese relative clauses, including both the structural probabilities of pre-relativizer constituents and the overall surface thematic orders in the relative clauses.This study investigates the comprehension of three kinds of subject-extracted relative clauses (SRs) in Mandarin Chinese: standard SRs, relative clauses involving the disposal  A robust asymmetry has been repeatedly reported in languages where the relative clauses follow the nouns they modify . In English, for example, relative clauses involving subject extractions like (1) have been found to be easier to comprehend than those involving object extractions like (2) Ford, . The hea(1) Subject-extracted relative clause:The composeri who __i adored the musician} drank a glass of wine.     {(2) Object-extracted relative clause:The musiciani who the composer adored __i} drank a glass of wine.     {Two main groups of theories have been adopted to account for this processing asymmetry, here referred to as “integration-based theories” and “experience-based theories.” The first group of theories focuses on the consumption of working memory in constructing filler-gap dependencies, suggesting that SRs in English are easier to comprehend (with shorter reading times and greater comprehension accuracies) because, relative to ORs, less working memory is required to process them. Within these integration-based theories, a number of proposals have been made as to precisely how the relevant processing costs are computed. A linearity account  than an object gap  thematic order in a language can be used as a perceptual strategy to facilitate sentence comprehension Bever, . AccordiThis brief summary highlights the fact that the overall advantageous reading of SRs in English is consistent with multiple theories of sentence comprehension though specific predictions about where the processing differences should be observed may differ. Gibson and Wu , for exaAn accumulating body of research over the past decade has painted a somewhat different picture of the processing of head-final relative clauses  sentences with relative clauses in (3) and (4)(3) Sentence with subject-extracted relative clause in Chinese:i aimu  yinyuejia  de    zuoqujiai    he-le         yi   bei     jiu      _i adore musician  rel composeri drink-asp  one glass wine      _      “The composer who adored the musician drank a glass of wine.”(4) Sentence with object-extracted relative clause in Chinese:i de   yinyuejiai he-le         yi   bei     jiu      zuoqujia    aimu  _i rel musiciani drink-asp one glass wine      composer adore _      “The musician whom the composer adored drank a glass of wine.”farther from the head noun  than object gaps, unlike English. These facts are illustrated in Figure Chinese displays a head-initial structure in verb phrases: like English, Chinese is a Subject-Verb-Object (SVO) language, with verbs preceding their NP object complements. At the same time, however, Chinese displays a head-final structure in NP: modifiers of nouns exclusively appear before the head noun. Because of this combination, while subject gaps in Chinese are higher and structurally closer to the complementizer/relativizer than object gaps (as in English), subject gaps are linearly Regarding gap-filler integration, therefore, the linearity account predicts that the gap-filler relation in a Chinese OR should be less taxing to construct than that in an SR. The structure-based account predicts the opposite: since fewer structural nodes intervene between the head noun and a subject gap, the dependency between these two should be easier to construct compared to one involving an object gap. Both accounts would predict the locus of processing differences on the head noun where gap-filler integrations take place.Regarding the effect of structural probabilities, given that SRs have higher frequencies than ORs in Chinese , and SRs with the long passive bei construction (henceforth “passive SRs”). Both the disposal ba construction and the passive bei construction involve functional morphemes that have been analyzed as light verbs or grammaticalized verbs in Mandarin Chinese. An example for each construction is given in (5–7). Sentences with relative clauses appear after referential contexts, which are intended to pragmatically motivate relative clauses so that the prenominal relative clauses are parsed as relative clauses when they appear in sentences.The present study addresses this methodological issue by holding the extraction site constant (only SRs) and investigating the processing of three different (5) Standard SR:i jiaoxing  furen de   zuoqujiai    he     yi    bei     jiu      __i wake.up lady   rel composeri drink one glass wine      __patient     agent      action               “The composer that woke up the lady drank a glass of wine.”bei) SR:(6) Long passive (i bei  furen   jiaoxing  de    zuoqujiai    he     yi    bei    jiu      __i BEI lady    wake.up rel  composeri drink one glass wine      __agent action            patient                        “The composer that was woken up by the lady drank a glass of wine.”ba) SR:(7) Disposal  between the gap and the filler. In terms of structural locality, since all three SRs involve extraction from subject position, the structural distance between the head noun and the gap are identical across all three structures (passing through two XP nodes—one CP and one IP), thus predicting no processing differences.The processing factors discussed above cast different effects on these three types of Chinese SRs. Let's first focus on the integration effects regarding the dependency between the subject gap and the head noun in each of the three structures, which are usually taken to be observable around the head noun region, where filler-gap integrations take place. In terms of linear locality Gibson, , the sambei indicates a missing subject NP that is to be connected with the object NP in the lower VP. Assuming that a relative clause parse has been adopted, the missing subject NP is taken to be connected both with the object NP and the head nounba also indicates a missing subject NP. Assuming again the processing of a relative clause, this missing subject NP would be taken as a subject gap connected with the head noun. An additional dependency in a disposal SR involving the displaced object NP after ba would add to the processing cost already incurred by the SR. The integration-based accounts, taking into consideration these additional dependencies, would then predict that both a passive SR and a disposal SR should be harder than a standard SR because (a) the former SRs involve additional dependencies, and (b) the dependencies in the former SRs are longer and more complex than that of a standard SR. Processing differences are expected to appear on and before the head noun.In addition to the gap-filler dependencies, the passive SRs and the disposal SRs involve additional displacement dependencies as depicted in Figures Next, we consider the overall structural complexity and structural frequencies involved in the three types of SRs. The standard SR is the simplest of the three constructions, as it contains the fewest number of structural layers and only has a single dependency relation (between the subject gap and the head). Passive SRs and disposal SRs are both more complicated in terms of the intricate dependency relations inside the VP/vPagent-action-patient), a passive SR should be the easiest to process among the three. Conversely, the thematic orders of standard SRs and disposal SRs are inconsistent with this dominant thematic order and should be more difficult to process than the passive relatives.On the other hand, the thematic order effect predicts different processing preferences. Since the surface thematic order of a passive SR matches the canonical thematic order in Chinese  vision, and were naïve to the purpose of the experiment. Participants gave informed consent to take part in the study. The study protocol was approved by Indiana University's Institutional Review Board.agent-action-patient. Following each context was a dialogue between two interlocutors, Xiaoming and Xiaomei, in which Xiaoming asks Xiaomei to identify one referent out of the two active referents, as in (9). Xiaomei's response starts with the target relative clause presented in a word-by-word moving window format. A sample of the experimental materials is given below:Twenty sets of sentences were included as the experimental trial, 16 of which were modified based on Gibson and Wu's  stimuli.(8) Context:     Yidong gongyuli   zhule fangdong yiji  liangge fangke     one       apartment lived landlord   and two      tenants     “A landlord and two tenants lived in an apartment.”     Yiwei zhuhu chaoxingle fangdong     one    tenant woke up    landlord     “One of the tenants woke up the landlord.”     Fangdong ze    chaoxingle lingyiwei zhuhu      landlord   then woke up    the other tenant     “The landlord woke up the other tenant.”Xiaoming: Wo tingshuo qizhong         yiming  zhuhu  hen  gao                 I     heard     among them   one      tenant  very tall                 “I heard one of the tenants was very tall.”                 Nayiwei    zhuhu hen  gao?                 which.one tenant very tall                 “Which tenant was very tall?”(9) Target sentence with a relative clause:     (i) Standard SRXiaomei: Chaoxing fangdong de    zhuhu           hen     gaorel  tenant          very    tall               woke up  landlord   V             N             REL Head Noun HN+1 HN+2                            “The tenant that woke up the landlord was very tall.”     (ii) Passive SRXiaomei: Bei     fangdong chaoxing de     zhuhu           hen      gaobei     landlord   woke up rel   tenant           very     tall              BEI   N             V            REL Head Noun  HN+1 HN+2                            “The tenant that was woken up by the landlord was very tall.”     (iii) Disposal SRXiaomei: Ba   fangdong  chaoxing de     zhuhu          hen      gaoba   landlord   woke up rel      tenant          very     tall              BA  N             V            REL Head Noun  HN+1 HN+2                            “The tenant that woke the landlord up was very tall.”Forty-eight additional sets of sentences following a similar format served as fillers. Sixteen of these fillers had relative clauses of various types in them; the remaining 32 fillers did not contain relative clauses. Altogether, 68 sets of contexts and sentences were pseudorandomly presented so that no two experimental trials appeared consecutively. Comprehension questions followed each trial to ensure that participants paid attention in reading the experimental materials. The words used in the relative clauses are provided in the Supplementary Materials.ba, and bei were presented in the same region as the following noun. After the last word, participants were given a true/false comprehension question focusing on the overall content of the context or the target sentence. Feedback was given if the participant's response was incorrect. Participants were instructed to read the sentences at a natural pace in order to answer the comprehension questions correctly. The reading time for each region, the time taken to answer the comprehension questions, and the responses to the comprehension questions were recorded. The whole experiment took an average of 40 min to complete.The experiment followed the standard moving-window self-paced reading design and was conducted using Linger 2.94 (developed by Doug Rohde)t-value of 2 is taken to be the threshold for statistical significance at α = 0.05. Question-accuracies were analyzed using generalized linear mixed models with a binomial link function. The dependent measures included comprehension accuracies (binary results), latencies in answering comprehension questions, and region-by-region reading times.Linear mixed-effects models treating both subjects and items as random effects were fit to both the comprehension accuracy data and the region-by-region reading time data using the lme4 package version 1.1-7 in R , 91.83% (standard SRs), and 86.28% . These results are summarized in Figure In terms of overall comprehension accuracy, passive SRs were comprehended more accurately than both standard SRs and disposal SRs. These results are consistent with the predictions of thematic order effect. Namely, passive SRs, whose thematic order followed the canonical thematic order, were comprehended with greater accuracies than both standard SRs and disposal SRs. No difference was found on the time taken to respond to the comprehension questions.Since the regions before and after the relativizers are hypothesized to be reflective of different processing effects, average reading times in the two pre-relativizer regions were compared to those in the post-relativizer regions (from the head noun to two regions after the head noun) across the three conditions. Figure In the pre-relativizer regions, passive SRs were read longer than standard SRs. In the post-relativizer regions, passive SRs were read faster than both the standard SRs and the disposal SRs. The reading time of each target region, including the two pre-relativizer regions, the relativizer, the head noun, and the two regions after the head noun, is further summarized in Figure Passive SRs were read longer than standard SRs in both regions inside the relative clause , and faster than disposal SRs from the second region in the prenominal clause to the head noun regionTo sum up, standard SRs were read with greatest ease in the earlier regions of the relative clauses. In contrast, in the regions following the relativizer, passive SRs were read more quickly than standard SRs and disposal SRs. The easier comprehension of standard SRs in the pre-relativizer regions is consistent both with integration effects  and with expectation-based constructional frequency effects . The easier comprehension of passive SRs in the post-relativizer regions, on the other hand, is only consistent with the prediction of thematic template mapping.The present study contrasted the reading patterns of three types of SRs in Chinese: standard SRs, passive SRs, and disposal SRs. Distinctive reading patterns were observed in the regions before and after the relativizer, suggesting the effects of different processing factors being operative. While the current experimental design intends to motivate relative clauses by using referential contexts, it is still unclear whether a relativized gap has indeed been postulated in the pre-relativizer regions given that a relative-clause parse is but one of several possible parses for the pre-relativizer regions. The structurally-ambiguous pre-relativizer regions showed reading patterns consistent with expectation-based theories of sentence comprehension  that matches the canonical order in a Chinese sentence and may be read with greater ease than those of a Chinese SR, whose pre-relativizer verb-noun sequence is non-canonical. In previous studies where relative clauses were not structurally disambiguated, greater processing costs were indeed associated with the pre-relativizer regions of an SR—an effect consistent with the prediction of structural probabilities as well as thematic orders  may be comprehended with greater ease. This thematic template effect may also be effective in the comprehension of SRs in English, whose surface thematic order matches the dominant thematic order in the language.The effect of thematic ordering on sentence comprehension can be understood as a processing heuristic used for efficiently coming up with thematic interpretations for sentences. The sentence processor keeps track of the linear positions of the content words in a sentence in forming thematic interpretations. The dominant thematic order of a language may serve as an “interpretation template,” to which the content words of a sentence are matched. The comprehension of sentences with more complex structures such as relative clauses can be facilitated by matching thematic orders against the dominant thematic templates. Since the dominant thematic template in Chinese is These effects of thematic order are in line with several existing theories of sentence processing. The idea of thematic templates has a similar flavor to Bever's  NVN heurIn conclusion, the reading time data for three sub-types of Chinese SRs reported in the present study supported two processes that are involved in the comprehension of Chinese relative clauses. Before reaching the relativizer, where the structure of the sentence is temporarily ambiguous, expectation-based incremental processing theories such as those of Hale , 2006 anThe critical evidence for the effect of thematic ordering comes from the easier processing of passive SRs, whose thematic role order conforms to the canonical thematic order of Chinese. Despite their more complex structural dependencies and lower constructional frequency compared with standard SRs, passive SRs were nevertheless comprehended with the greatest accuracy and processed with the shortest reading times in the post-relativizer regions. The current study therefore suggests that the comprehension of relative clauses in Chinese is sensitive to both the structural probabilities of constituents as well as the thematic orders involved in the relative clauses. In our effort to understand relative clause comprehension, it is important to take both of these factors into account.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools. Under conditions not fully understood, it changes into the amyloid or Scrapie (PrPSc) form  prion. In its soluble form, Sup35 participates in translational termination but is unable to do so in its aggregated form. Sup35 plays a role equivalent to the bacterial release factor 3 and thus, translation termination depends on fully functioning soluble Sup35. The aggregation state of Sup35 can be monitored by translation of the  hazards ,21. BesiCompounds identified by Sup35 screening are tested further in another screen based on URE3 prion system, which uses the Ure2 protein . Since SHuge libraries of compounds have been tested with the yeast-prion based colorimetric assay and several compounds have been identified to be active against prions. Most of them are organic compounds belonging to family kastellpaolitines , phenantetc.; the suffix indicates their inactivity in prion assay  phenotype in the yeast-prion based assay. The inactive derivatives are named 6APi, GAi on assay . The anton assay   was developed. Affinity matrices were constructed with 6AP and GA chemically linked to the resin via a linker, so that they could physically interact with the target molecules ,25. The to PrPSc . This isThe results of the pulled-down assay led to another important observation. The Western blot did not show bands corresponding to the prion proteins, but did reveal bands corresponding to several ribosomal proteins. This would be possible only if 6AP and GA would target rRNA and consequently pull-down the whole ribosome from the 6AP-/GA beads. To test that crude extracts of [PSI+] yeast were treated with RNase and then affinity chromatography was conducted as described. As expected, no bands corresponding to the ribosomal proteins could be seen in the Western blot. This result confirmed that the binding of ribosomal proteins to the 6AP/GA is rRNA-dependent and ribosome is the cellular target of 6AP and GA. Thus, 6AP and GA inhibit prion formation in an indirect pathway.et al. who found that denatured proteins could be refolded to their functionally active form by including ribosomes in the refolding buffer . T. Tin vits et al. . in vitro . T. Tin vitPrimer extension studies were also done to identify the binding sites of the protein substrates on the domain V rRNA. For that, three unrelated substrate proteins , freshly diluted from denaturation mix, were UV cross-linked to the domain V rRNA . InteresE. coli 23S rRNA) and eukaryotic (Saccharomyces cerevisae 25S rRNA) domain V rRNA were subjected to mutagenesis on the homologous sites. For comparison, mutations were also introduced in some random sites on domain V rRNA. All mutated domain V rRNAs were tested for their protein folding activity in PFAR assay. In parallel, some of the mutated domain V rRNAs were UV cross-linked with 6AP and human carbonic anhydrase (HCA) as a protein substrate (in separate reactions) and were checked by primer-extension. To study the effect of specific nucleotides on PFAR, mutations were introduced at the common 6AP- and protein binding sites on the domain V rRNA . Both thE. coli domain V rRNA. Corresponding mutations in the eukaryotic domain V rRNA also showed highly defective protein folding. Control mutations at residues 2486–2488 on 23S rRNA and equivalent positions on 25S rRNA, which did not correspond to protein or 6AP binding sites, showed no defect in PFAR. Most of the altered domain V rRNAs with mutation in 6AP- or protein binding sites were defective in PFAR . The bigWhen the mutant domain V rRNAs defective in PFAR were tested by UV-crosslinking followed by primer extension analysis, distinct changes in the binding maps of both HCA and 6AP were noticed . For theMK) increases, but the maximal rate of the reaction (catk) remains constant. Furthermore, the inhibition of PFAR by 6AP and GA can be reversed by addition of RFMs in excess. These data confirm that 6AP and GA inhibit PFAR in a competitive manner. 6AP and GA occupy the binding sites on domain V rRNA, which also constitute the active site for PFAR. When the active sites are blocked with 6AP/GA, the protein substrates fail to bind to domain V rRNA and fold in self-folding pathway. Since 6AP/GA do not interact with the protein substrate, they do not affect self-folding of the proteins. It is likely that the domain V rRNA and protein substrate interactions are transient and the protein actually folds to the active state after being released from the RFMs. This explains why the kinetics of refolding of HCA, monitored by measuring its enzymatic activity, is not affected by the presence of RFMs or the PFAR inhibiting antiprion compounds [As shown in ompounds ,46.3 decreases gradually upon addition of domain V ribosomal RNA , as seen for the antiprion activity of the compounds and also for PFAR inhibition. This reconfirms that 6AP and its derivatives inhibit PFAR by binding to domain V rRNA and that PFAR is implicated in prion formation or propagation. In parallel with the UV cross-linking studies, the intrinsic fluorescence of 6AP derivatives has been used to study their binding to domain V rRNA . This isomal RNA A. FluoreA at all A, inset.rescence B. The eqectively , which iDensity functional theory (DFT) calculations have been used to explain the differential binding behavior of 6AP and its derivatives towards domain V rRNA . Nucleus3 and 6APi are listed in 3) groups at the 8 position in 6AP8Cl and 6AP8CF3 does not affect the planarity of the phenanthridine ring. In fact, these substitutions enhance the aromatic ring current and hence, these two 6AP derivatives are better binders of domain V rRNA than 6AP. However, substitution of the amine nitrogen of 6AP with a 2-(butan-1-ol) group makes 6APi nonplanar. Moreover, the bulky substitution extends from the plane of the phenanthridine ring, which could create steric hindrance for binding. Thus, the deviation of planarity in 6APi and the steric hindrance due to the bulky substitution inhibits 6APi from binding to domain V rRNA  work in blocking prion processes. These antiprion compounds are primarily rRNA dependent inhibitors of PFAR. When these compounds inhibit PFAR by binding to domain V rRNA, the prion proteins probably fold into the unstructured aggregates and get degraded by cellular proteases, thereby blocking prion formation/propagation. The current view of PFAR and its role in prion processes is illustrated in


This permits smaller capacitors in memory devices and is a step forward in making ferroelectric domain-engineered nano-electronics.The dielectric permittivity in ferroelectric thin films is generally orders of magnitude smaller than in their bulk. Here, we discover a way of increasing dielectric constants in ferroelectric thin films by ca. 500% by synchronizing the pulsed switching fields with the intrinsic switching time (nucleation of domain plus forward growth from cathode to anode). In a 170-nm lead zirconate titanate thin film with an average grain size of 850 nm this produces a dielectric constant of 8200 with the maximum nucleus density of 3.8 μm High-dielectric ferroelectric thin-films are of great importance for nanoelectronics, where their capacitance should be maximum to reduce size and power consumption. The capacitance can be enhanced by making the films thinner, but that is limited by breakdown. An attractive alternative would be to increase the dielectric constant. An increase of an order of magnitude would permit a smaller “footprint” of areal size on the chip; since 90% of the area of such a chip is capacitor, with smaller resistors and transistors taking up only ca. 10%, this would permit a 90% overall size reduction. Lead zirconate titanate (PZT) thin films could be a viable option for such dielectric material having a very high dielectric constant. In this work, the enhancement of dielectric constant in ferroelectric PZT thin films from a normal value of ca. 800 to 8,200 is reported. This increase was realized via ac-voltage drive synchronized to the anode-cathode transit time for domain wall motion. By reversing the applied electric field just before the nucleated reverse domains penetrate through interfacial electrode-dielectric (“dead”) layer to reach the opposite electrode3/BaTiO3/SrRuO3 sandwiches, and the related functionalities in these devices can be achieved in the ns-ps time scale as their physical dimensions shrink down into the nanometer scale2345Pf) can be reversibly switched to follow the external stimuli of an alternating-current (AC) field (Ef). However, the experimental value of the dielectric constant is always much smaller than the expected value  because much of the polarization charge does not follow the small oscillating AC field. Therefore, these effects have severely hampered the applications of ferroelectrics to memory devices, miniaturized sensors, actuators, phase shift antenna arrays, and energy harvesting systems7Classic ferroelectric oxide films provide large ionic displacements of individual atoms down to the atomic layer thickness, for example, ~2.4 nm for SrRuO3 thin filmsIt has also been known since the 1980s91011121314151617193 single crystals with hetero-valence impurities a large nonlinear electrostriction is generated during 90° domain switching2324V) is applied. This figure implicitly assumes a single crystalline film, but the same model can be applied to coarse-grained polycrystalline films when the interference effect from the presence of grain boundaries is weak. It is assumed that the down-polarized domains at time t0 have residual back-switched clusters or “nuclei” even in the upward pre-poled state , Nucleus 1 is assumed to grow rapidly and to form a fully switched domain during the interval time of Δt, whereas Nucleus 2 is still penetrating the film thickness  1 remains unchanged, but Domain (Nucleus) 2 shrinks back quickly and releases the polarization charge, Pnu (right panel in t = t0 + 2ΔtIn BaTiO0.2Ti0.8)O3 thin films, where the embedded in-situ piezoresponse force microscopy (PFM) probes induced triangular domain nucleation with a wall that was inclined by 55° toward the substrateExperimental evidence of the reversible nucleating domain growth and the subsequent sideways wall motion has been found in the cross-sectional transmission electron microscopy observation of non-penetrating triangular domains in epitaxial (001) Pb must be estimated. This can be done by understanding that Δt is the time needed for the switching current flows to induce Pnu, with the current density of6Under an AC driving field, the nucleating domains can oscillate longitudinally and thus contribute to the nonlinear capacitance through the periodic domain expansion and contraction. To achieve this goal, the contribution of Vc,n and S are the coercive voltage for domain nucleation and the electrode area, respectively. Thus:where J was measured as a function of inverse of coercive field 0.4Ti0.6)O3(PZT)/Pt polycrystalline thin film capacitors with a 170-nm PZT thickness and an average PZT grain size of ~800 nm were deposited on TiOx/SiO2/Si substrates  solid line in J (or V).J and Δt functions, the ferroelectric capacitance-voltage (Cf–Vf) loops for a 100 × 100-μm2-area capacitor can be directly estimated using a delta pulse technique . At each voltage the nonlinear capacitance was measured from the difference between the two discharging capacitor charges at the two voltages of Vf + ΔVf and Vf , as follows:Using the calculated Qf is the capacitor-accumulated charge per area and Vf is the voltage across the ferroelectric layer under V in the RC circuit. When the pulse time is short enough to keep the domains within the film thickness at a given Vf, the capacitance is dominated by the reversibly oscillating domains. However, this type of measurement does not necessarily exclude the contribution from the polarization switching charge when the sweeping voltage changes by ΔVstep, especially when the polarity of Vf becomes opposite to the pre-poling voltage direction. To alleviate this problem the capacitor was repeatedly stressed at each value of Vf with a unipolar pulse width of 250 ns for 70 cycles, where the net voltage drop across the film iswhere V and V − ΔVstep  and Qf (Vf + ΔVf) were measured sequentially by superimposing ΔV < 0 above V  and Qf(Vf) was estimated; this was attributed to the shrinking domain charges (Pnu) and normal dielectric capacitor charges of both unswitched (or yet-to-be-switched) and switched regions. Finally, the complete Cf –Vf loops with increasing and later decreasing Vf values (−5 V → 5 V → −5 V) were measured under the different stimulating AC voltages of ΔVf (from −0.01 to −0.05 V). The results are summarized in ε reached a maximum of 8,200 when ΔVf = −0.01 V, which is the largest dielectric permittivity ever reported from ferroelectric thin films  temperatures and/or morphotropic phase boundaries, which should be below the order of 630 estimated from the measurements in a long mismatched time of Δt = 100 ms  nonlinearity (or the wide Pnu distribution) near the coercive voltage  for reverse domain nucleation  τ was increased in a stepwise manner from 5 to 500 ns which were applied to the top electrode of a 100 × 100 μm2-area capacitor (J(t)] as well as their transformation into Pf-Vf hysteresis loops  were measured using an oscilloscope, which was serially connected to the sample with the total internal resistance of R = 100 Ω at ambient temperature. t = τ can be obtained from Eq. J(t) the capacitor discharge and domain switching charge can be calculated as functions of Vf. To calculate the discharge free from the influence of the irreversible domain switching charge, the dissipating current after t > τ was integrated, and the results were plotted as functions of Vf for different V values, as shown in V values that were anti-parallel P) can also be estimated from the integration of the entire J (t) curve (0 < t < ∞) for each Vf , as it comprises domain switching and capacitor charging/discharging. The results are shown in V values (size = 104 μm2) and sizes (V = 3.5 V). In J(t) up to ~50 ns are attributed to pure capacitor charging and reverse domain nucleation. The subsequent constant J(t) up to ~200 ns corresponds to the sideways wall motion of the domain growth (to be discussed later). Then, the current decays exponentially with time as the domain switching is completed, and Vf eventually reaches V. Also shown in Pnu, as functions of time. When the same measurement was performed with a voltage whose polarity was parallel to the pre-poling direction, the J(t) and Vf if there is no contribution from other factors). There were two more critical observations: (1) all the V = 2–5 V showed sharp peaks near the coercive voltages for the reverse domain nucleation , as indicated by the (pink) arrows in Vf graphs; and (2) Vf) equals Vf  + Vimp) for |Vf | > |Vc,n|, which suggests that the difference between Vimp. The sharp peaks at Vc,n in V was turned off. This means that some parts of the domain inversion were not completed throughout the film thickness during the period of the application of V. Therefore, the full nucleation charge Pnu, which was discharged when V was turned off, can be defined. This corresponds to the discharge related to the rapidly shrinking reverse domains, as represented by Domain 2 in Vc,n that differ from the coercive voltage  for sideways wall motion can be estimated from the sharp peak position in Vc,s corresponds to the voltage at which half of the irreversible domain reversal is completed, which is usually regarded as the coercive voltage of a ferroelectric capacitor in the measurement of the polarization-voltage (Pf –Vf) hysteresis loop. The presence of a hump near t ~ 70 ns in the t plot in t ~ 80–150 ns). It is quite natural that these Pnu peaks were not observed in the Vf graphs, as there was no reverse domain nucleation and growth. The imprint field is believed to be built in by the interfacial trapped charges during the pre-polingAlthough the large apacitor . The curPnu and to understand its influence on ε of the PZT layer, the following points were considered: It was noted that Vf) in Pnu(Vf)]; (2) the charge density related to the non-switched (or yet-to-be-switched) regions [the red region in Vf)]; and (3) the charge density related to the irreversibly switched regions [Domain 1 in Vf  + Vimp)]. The volume fraction of the switched domain, x, at a given Vf can be estimated from Ps is the saturation polarization inferred from the saturated Pf –Vf hysteresis loop (Vf) is the sum of Pnu(Vf), [1 − x(Vf)]Vf) and x(Vf)Vf  + Vimp). Vf) could be represented by V is large enough to induce all the needed charging. ΔP(Vf), which is needed to calculate x at each Vf , is already given in To estimate quantitatively sis loop . TherefoPnu can be calculated as a function of Vf for different V and S values (Pnu(Vf) curve has a sharp peak near Vf that coincides with the onset of an abrupt increase in |ΔP|. When Vf at which half of the |ΔP| increase is obtained is taken as Vc,s for the domain sideways wall motion, |Vf| for the peaked Pnu is slightly lower than |Vc,s|. Such Vf for the peaked Pnu can define the coercive voltage for reverse domain nucleation : it is notable that |Vc,n| is always slightly lower than |Vc,s| for sideways wall motion for different V and S values . This value slightly increases up to 4.4 μC/cm2 at 77.6 K −2 from this experiment with macroscopic capacitors  is comparable to the values of 4.4 μm−2 (area: 1.5 × 1.5 μm2) in ref. −2 (area: 3 × 3 μm2) in ref. et al., using focused X-ray microdiffractionwhere 2 at Vc,n due to the non-penetrating reverse domain nucleation was quantitatively estimated. The restoring force (imprint voltage effect) was large enough to shrink the nucleating domains when the anti-parallel excitatory voltage was removed, which opens the door for the development of new domain engineering. A huge dielectric permittivity  from the periodic domain oscillations was estimated, which was free from the interfacial “dead-layer” effects even in ultra-thin films. This longitudinal oscillation differs from the transverse oscillation of fully penetrating walls91011121314151618193435The polarization oscillations of the non-penetrating domains within the thickness of ferroelectric PZT thin films 170 nm) were observed under a well-controlled AC pulse field. With the help of the short-time imprint effect, the maximum reversible polarization of ~4.1 μC/cm0 nm wereivity ~8,0 from th0.4Ti0.6)O3 thin films were synthesized through sol-gel spin coating on Pt/TiOx/SiO2/Si substrates with a methoxyethanol-based precursor solution2-Pt electrodes were sputtered on the films as the bottom and top electrodes (See Pnu(Vf) distribution, all the samples were first stressed at ±6 V with suitable bipolar pulse widths for 104 cycles and then aged at ambient temperature for more than a week, to establish the same imprint history for all the random domains. The electrical measurements were carried out at both ambient and cryogenic (77.6 K) temperatures.Polycrystalline Pb.


To study the relationship between amplitude of spontaneous retinal venous pulsatility (SRVP) and retinal nerve fibre layer (RNFL) thickness in glaucomatous eyes, and to determine if this parameter may be a potential marker for glaucoma severity.85 subjects including 50 glaucoma  and 35 normals  were studied. SRVP amplitude was measured using the Dynamic Vessel Analyser  at four regions of the retina simultaneously within one disc diameter from the optic disc—temporal-superior (TS), nasal-superior (NS), temporal-inferior (TI) and nasal-inferior (NI)). This was followed by RNFL thickness measurement using spectral domain optical coherence tomography . The correlation between SRVP amplitude and corresponding sectoral RNFL thickness was assessed by means of non-linear regression (i.e. logarithmic). Linear regression was also applied and slopes were compared using analysis of covariance (ANCOVA).(p>0.05). Since the slopes are not significantly different, it is possible to calculate one slope for all the data. The pooled slope equals 10.8 (i.e. RNFL = 10.8SRVP+41).Greater SRVP amplitude was associated with thicker RNFL. Global SRVP amplitude was significantly lower in glaucoma eyes compared with normals (p<0.0001). The correlation coefficient of the linear regression between RNFL and SRVP at TS, NS, TI and NI quadrants in the glaucoma group were r = 0.5, 0.5, 0.48, 0.62. Mean SRVP amplitude and RNFL thickness for TS, NS, TI and NI quadrants were 4.3±1.5, 3.5±1.3, 4.7±1.6, 3.1±1 μm and 96±30, 75±22, 89±35 and 88±30 μm, respectively. The ANCOVA test showed that the slope of linear regression between the four quadrants was not significant While SRVP was present and measurable in all individuals, the amplitude of SRVP is reduced in glaucoma with increasing RNFL loss. Our findings suggest the degree of SRVP may be an additional marker for glaucoma severity. Further studies are needed to determine the mechanism of reduction in SRVP, and whether changes can predict increased risk of progression. Spontaneous retinal venous pulsations (SRVP) result from an interaction between intraocular pressure (IOP), retinal venous pressure (RVP) and cerebrospinal fluid pressure (CSFp). With elevated CSFp, raised RVP or reduced IOP, the intravascular pressure gradient across the prelaminar and retrolaminar portions of the central retinal vein decreases, leading to cessation of SRVP–3. MorgaDifferent studies have investigated the nature of SRVPs,13. It wThe purpose of this study, was to investigate the relationship between amplitude of SRVP and RNFL thickness at four regions , nasal-superior (NS), temporal-inferior (TI) and nasal-inferior (NI)) of the retina in glaucomatous and normal eyes. We used RNFL thickness to stage severity of glaucoma in those sectors and amplitude of pulsations to give a quantitative measure of pulsation in all subjects.50 open angle glaucoma patients  and 35 healthy volunteers  were included in the study. All subjects went under a series of ophthalmic tests including slit lamp examination, IOP measurement (Goldman tonometry), measurement of SRVP amplitude using the Dynamic Vessel Analyzer  and RNFL thickness measurement using Spectralis Optical Coherence Tomography . A 75D lens was used to observe the presence or absence of SRVP prior to DVA measurements.Glaucoma subjects were identified as having definite glaucomatous changes in the neuro-retinal rim, 3 patients did not yet have visual field defects, while the remaining 47 subjects had corresponding visual field changes with MD range 0–16 dB. A past history of acute angle closure, retinal vein occlusion or other retinal pathology was an exclusion. Normals were included if they had no history of eye disease, a normal fundus on ophthalmoscopy with no visible vascular changes. All subjects had no symptoms or signs of raised CSFp, and no history of diabetes.Pupils of all subjects were dilated 20 minutes prior to the tests using Tropicamide 1% . SRVP was measured at four sites within 1 disc diameter from optic disc , nasal-superior (NS), temporal-inferior (TI) and nasal-inferior (NI)) for 100 sec at a 25 Hz sampling frequency . From thOCT scans were taken at 30° field of view centred on the optic disc. The Spectralis built-in software was used to segment the OCT B-scans and measure RNFL thickness at the relevant four sites .To evaluate reproducibility and potential fluctuation, 20 of 50 glaucoma patients were selected randomly and re-tested 6 months after their initial visit. This study was performed in accordance with the guidelines of the Tenants of Helsinki and approved by the Macquarie Universities’ ethics committee. Written consent was obtained from the subjects after explanation of the nature and possible consequences of the study.The average SRVP amplitude and RNFL thickness between glaucoma and normal controls were compared using student’s t-test. Linear regression was then used to assess the relationship between SRVP amplitude and RNFL thickness at four quadrants of the optic disc. The RNFL distribution has been reported in many studies and structure /function maps developed ,17. SincWe also performed correlations between the mean of the two superior and two inferior SRVP amplitudes with RNFL mean thickness using the two superior sectors (TS and NS) and the two inferior sectors (TI and NI) see . FurtherAnalysis of covariance (ANCOVA) was used to compare the slope of the linear regressions. One way Analysis of Variance (ANOVA) was used to measure the difference in the linear regression slope between SRVP amplitude –RNFL thickness across the four regions in glaucomatous eyes. Statistical analyses were performed using Graphpad Prism 6 .Data for all 85 subjects was analysed to determine topographical correlation by individual sectors between SRVP and local RNFL thickness. The global SRVP amplitude  was significantly lower in the glaucoma group compared with the normal controls . Similarly, global RNFL thickness was significantly lower in the glaucoma group compared with the normal controls  .When individual sectors of the nerve fibre layer thickness around the optic nerve head were analysed for correlations, we found a positive correlation between SRVP amplitude with RNFL thickness in all four sectors. Linear regression was applied to determine the association between SRVP amplitude and RNFL thickness at four sectors of the retina in glaucoma and normals. The last 2 rows of 2 (goodness of fit) of 0.35for superior and 0.3 for inferior sectors. To verify whether the correlation holds for average RNFL thickness on the temporal ornasal sector with the superior and inferior SRVP amplitude on the same side of the optic disc, we correlated mean RNFL for all the temporal sectors with mean SRVP for the two temporal veins (superior and inferior). Results showed that the correlation was no stronger than for individual sectors at r = 0.48. Similarly the correlating nasal three sectors of RNFL including both superior and inferior nasal vessels yielded r = 0.5. he reason for analysing this separately was that there is no absolute demarcation between vascular beds and corresponding RNFL distribution.Analysis of Covariance (ANCOVA) was used to study the significance of the linear regression slopes applied to the RNFL-SRVP relation in the four quadrants. To assess any long-term fluctuation the mean superior and inferior SRVP amplitude and RNFL thickness for baseline and 6 month follow up are shown in In this study we investigated the relationship between nerve fibre layer thickness and retinal venous pulsatility. We investigated glaucoma subjects and normal controls. We found a significant difference (p<0.0001) between the two groups with lower SRVP amplitude in glaucoma. However, the relation also held in the controls despite a narrower range of RNFL thickness. When analysis by sectors was performed, we observed lower SRVP amplitudes in those sectors with greater RNFL loss. It has been reported that ocular blood flow is reduced in glaucoma –20. Ther2 = 0.43). A more recent study investigated the relationship between retinal blood flow and RNFL thickness[Plange et al showed athickness. An inteReduction of blood flow could be due to an increased venous resistance in glaucoma subjects . A reducWhile the generation of SRVPs is not solely dependent on blood flow, reduced ocular blood flow and average venous diameter might contribute the finding of lower SRVP in regions of RNFL loss. We cannot determine from this study whether the reduction of SRVP is a primary event involved in the mechanism of glaucomatous damage , or secondary to reduced blood flow, or a combination of both factors.Alteration of SRVP amplitude in glaucoma may also be influenced by other parameters such as thinning of lamina cribrosa and an increase in the trans-laminar pressure gradient. Jonas et al showed that the lamina cribrosa was significantly thinner in glaucoma patients compared with normals (p<0.001) and that the distance between intraocular space and the cerebrospinal fluid (CSF) space was significantly shorter (p<0.001). PreviouIdentification of SRVP clinically is a very subjective phenomenon and is influenced by microsaccades during observation, However this study is not reporting on visible SRVPs at the optic nerve head, but in the adjacent retinal veins beyond the neuroretinal rim. Our previous study established the waveform of these pulsations was identical to that recorded on the disc itself. ClinicaIn this study we did not measure blood pressure and heart rate, however our previous studies have shown that blood pressure variations do not affect SRVP amplitude . We are We acknowledge that the distribution of nerve fibres originating in each of the optic disc sectors as defined by the OCT RNFL analysis does not exactly match the venous drainage distribution for the same vessel in that sector. We analysed various combinations of sectors and report here the best correlated sectors, which occurred when individual sectors were compared (eg TS RNFL correlated with the TS vein), or when both superior or both inferior veins were averaged and compared with the two corresponding RNFL sectors for superior or inferior RNFL.Previous studies have shown acceptable reproducibility coefficients for the DVA, ranging from 1.3% to 5%, with reproducibility being slightly higher for retinal veins. In our While a lack of SRVP has been reported as a risk factor for glaucoma, other factors such as raised CSFp or lower IOP are also known to decrease SRVP amplitude. While we do not know the actual CSFp in our subjects, the IOP was normal in the controls, and in the glaucoma subjects IOP was under treatment and was in the normal range. We also do not think this is a drug effect directly on the vessel walls, since our previous study in normals of the acute effects of an alpha agonist and a beta blocker on SRVP did not show vascular effects. FurtherIn conclusion, SRVP amplitude shows a positive correlation with RNFL thickness in both glaucoma subjects and normal controls. is means that SRVP amplitude is reduced in areas where nerve fibre layer loss has occurred in glaucoma. This is more evident on the temporal veins where the nerve fibre loss is more dominant in glaucoma patients.


These proteins, which undergo domain swapping, have acquired much attention owing to their involvement in human diseases, such as conformational diseases, amyloidosis, serpinopathies, proteionopathies etc. Early realisation of proteins in the whole human genome that retain tendency to domain swap will enable many aspects of disease control management. Predictive models were developed by using machine learning approaches with an average accuracy of 78%  to predict putative domain swapping in protein sequences. These models were applied to many complete genomes with special emphasis on the human genome. Nearly 44% of the protein sequences in the human genome were predicted positive for domain swapping. Enrichment analysis was performed on the positively predicted sequences from human genome for their domain distribution, disease association and functional importance based on Gene Ontology (GO). Enrichment analysis was also performed to infer a better understanding of the functional importance of these sequences. Finally, we developed hinge region prediction, in the given putative domain swapped sequence, by using important physicochemical properties of amino acids. Computational methods for classification, annotation and prediction of biologically important questions are rapidly improving our knowledge of protein sequence-structure-function relationships. Detailed analysis of such relationships of protein improves our understanding of sequence features and its role in different biological pathways and diseases. Understanding the mechanism of protein oligomerization can influence many aspects of protein research such as drug discovery. This will also add to the existing pool of knowledge regarding amyloid formation and aggregation-related diseases.3D-domain swapping is one of the mechanisms of protein oligomerization and is also known to be involved in protein aggregation processes. 3D-domain swapping was first reported as a mechanism of oligomerisation in RNase A by Crestfield and coworkers n 1962 after lyophilization in 50% acetic acid .Three classes of domain swapping viz., bonafide domain swapping (BDS), quasi-domain swapping (QDS) and candidates for 3D-domain swapping (CDS) have been identified by Eisenberg and others in 2002 . Most im3D-domain swapping is further classified on the basis of position of the swapping domain in the protein structure into three groups viz., N-terminal domain swapping, C-terminal domain swapping and the less common central domain swapping.Collection of such protein structures which are involved in 3D-domain swapping in the form of database is of great help to researchers for further analysis and detailed study of this phenomenon . To dateIn recent times, various computational and experimental studies were employed to understand the mechanism of 3D-domain swapping in detail. Sequence and structural features were used to predict 3D-domain swapping from protein sequence earlier . 3D-domaHere, we present a computational approach towards prediction and genome-wide analysis of 3D-domain swap protein from mere sequence information by using machine learning approaches such as Random Forest (RF)  and suppDue to diverse structural, functional, structural and pharmaceutical implications, the prediction of proteins with a potential to engage in 3D-domain swapping from mere sequence information would be a great advantage in identifying plausible drug targets for therapeutic control of such diseases. Technical limitations of structural elucidation of proteins at such high number, and in higher oligomeric conformations by using crystallography or NMR experiments set the need to develop computational methods. In this work, we have restricted our feature extraction only to sequence space and it is the first attempt of this kind. Finally, we predict hinge regions for the domain-swapped proteins by using a sliding window approach based on the peculiar physicochemical features of residues in the hinge region.3D-domain swapped protein molecules are often associated with aggregation diseases or proteinopathies in humans. Till now, no comprehensive study has been reported to analyze proteins involved in 3D-domain swapping from a genome-wide perspective. We have performed the initial investigation of putative 3D-domain swapped proteins at the level of protein domains, Gene Ontology, KEGG pathways and Disease Ontology.Training was performed on a dataset containing 2000 protein sequences comprising of positive (1000) and negative (1000) datasets. We have performed five-fold cross validation on testing dataset. The RF prediction model achieved an accuracy of 81.7% with 81.5% of sensitivity, 81.8% of specificity and a MCC value of 0.64. The SVM prediction model achieved an accuracy of 73.9% with 61.9% of sensitivity, 85.9% of specificity and MCC value of 0.61 . We havePerformance of prediction using an independent validation dataset of the well-known cases of 3D-domain swapping from 3DSwap database resulted in 100% accuracy . For anoFurther analysis of these sequences reveals that 27 out of 99 sequences (3D-domain swapped predicted sequences) are enzymes and 12 of them contain kinase domains. We also analyzed the domain distribution of these positively predicted sequences . Pkc likO. tenuiflorum to maximum of 44.2% for Homo sapiens (considering only reviewed genome sequences) , M. truncatula (20.9%), S. tumerosum (36.5%), S. lycopersicum (25.5%), and O. tenuiflorum (15.5%). Non-plant genomes on which these prediction models were applied are M. tuberculosis (28.2%), B. thuriensis (43.3%) and H. sapiens (44.2%). The numbers in brackets represent percentage of consensus positive domain swapping sequences.RF and SVM models were applied on five plant genomes and three non-plant genomes including the human genome, for prediction of 3D-domain swapping. Common prediction results from both the models range from a minimum of 15.5% in quences) . ConsensWe have performed 3D-domain swapping prediction on gene products in the human genome for reviewed and unreviewed entries, separately. Out of 20,247 reviewed entries in the human proteome, 57% of these  are predicted as 3D-domain swapped by RF model and 61%  sequences are predicted as positive by SVM method, thereby identifying 44% of the sequences which were predicted positive by both the methods. In case of the entire set (reviewed and unreviewed) consisting of 115,830 sequences, 24% of these are predicted positive for domain swapping.In the case of sequences positively predicted sequences by both RF and SVM methods (44%), a detailed analysis of the presence of domains was carried out and 2346 domain architectures were observed. Domain distributions of these sequences were also examined and it was observed that protein kinases, Immunoglobulin-like domain and cadherin domains are most populated . Other pFurther, these positively predicted sequences were enriched in 254 metabolic pathways. Protein ubiquitination and protein glycosylation pathways have a maximum number of sequences from 3D-domain swap predicted dataset . Top enrhttp://caps.ncbs.res.in/download/Human_pred/reactome/For the disease association and biological pathway of these positively predicted sequences, Reactome database was also consulted. The major diseases associated with the predicted sequences are classified into 18 groups , few of . Some of the case study examples chosen are seminal ribonuclease (11BA), promyelocytic leukemia Zinc finger protein PLZF (1BUO) and SH3 Domain (1AOJ).On the basis of experimentally reported hinge regions and swapped region of domain swapped protein structures, we found that the average hinge region is of 6.7 residues (ranging from one to 66 residues). A similar analysis was also performed on the length distribution of swapped domain in the known cases of domain swapping. Swapped domains are of an average size of 26 residues, ranging from 5 to 316 residues. Well-known examples of domain swapping were used for the study of hinge regions and were mapped back to structures as case study . Some ofSeminal ribonuclease retains an α+β fold, where the N-terminal region is engaged in 3D-domain swapping. This protein belongs to pancreatic ribonuclease domain family by Pfam database. Similarly, promyelocytic leukemia Zinc finger also belongs to α-and-β class and POZ fold according to SCOP database. It is a homodimer with N-terminal type of 3D-domain swapping. The normalized score  of human genome. Prediction results of known examples of 3DSwap database were mapped on sequences based on cumulative maximum scores to continuous three residues. The regions with maximum score were called as first level of prediction. On the basis of these scores other putative hinges are also demarcated. For the first level, coverage of 67.8% was achieved, when the predicted best hinge was considered for calculation. When the second best hinge region on the protein sequence was also included, along with the best hinge region, a coverage of 71.3% was achieved.etc. in such molecules from one case to another. Besides these deviations, several other features arise, such as incomplete data (e.g. absence of coordinates of residues for significant region in protein structure), central loop swapped entries, small hinge region, domain swapped oligomer with elaborate interface, intertwined domain swapped proteins and multiple hinge regions pose severe challenges and were hard to address considering small frequency of occurrence. Small hinge regions, generally less than five residues, are difficult to predict since the cumulative scores could result in false positives  of sequences have more than or equal to 50% disordered regions. The result shows that most of the sequences are well structured as found in globular proteins.Early identification of 3D-domain swap proteins on genome scale would help in better management of disease caused by protein aggregation and other protein domain swap related phenomena. Machine learning approaches, classifiers (RF and SVM models) have been developed to predict domain swapping at the genome level from mere protein sequence information. An accuracy of 81% and 74% were achieved for the two methods, respectively. These prediction models were applied to several complete genomes with a special emphasis on the human genome. To our knowledge, this is the first attempt to predict domain swapping at the genome level. Almost 44% of the sequences of the human genome were predicted as putatively involved in domain swapping. We observed the same trend when we applied these two models to different genomes.Functional annotation of all the positively predicted sequences from the human genome were carried out in terms of their distribution in different Pfam protein families, Gene Ontology, Biological pathways and Disease associations. These positively predicted sequences were dispersed in many Pfam protein families with different Gene Ontology annotations and in several pathways with substantial disease associations. Our approach helped us to understand enriched protein domains, Gene Ontology terms, biological pathways and Disease Ontology in putatively predicted protein sequences and their role in mediating various human diseases. Protein kinases, immunoglobulin-like domains and cadherin domains are most populated domain families observed in this dataset of sequences positively predicted for domain swapping. Few of the highly enriched human diseases in the positively-predicted dataset are, abnormal metabolism in phenylketonuria, defects in vitamin and cofactor metabolism, signaling by EGFR in cancer etc. The gene ontology terms for cation channel activity, metal ion transmembrane transporter activity, gated channel activity, ion channel activity, ATPase activity etc. have maximum fold enrichment under the biochemical activities.We have also developed a hinge region prediction tool, mainly based on important physicochemical properties of the residues in the hinge region. Further distribution of these molecules across structure, sequence and function families shows generic nature of this mechanism emphasizing the fact that this mechanism occurs independent of sequence, structure and function.Hinge region prediction was performed based on the important features of amino acids present in this region. This prediction tool is applied to all the predicted (8945) 3D-domain swapped sequences from human genome. Few of the peculiar features considered in this study are as (a) residues in the hinge region prefer to be small, but not hydrophobic or aliphatic in nature. (b) They are found less often in α-helices, and more often in turns or random coils. (c) Active site residues were found to coincide significantly with hinges. (d) Hinges are also more likely to occur on the protein surfaces than in core. (e) Propensity of amino acid residues in the hinges. (f) Hinge regions in domain swapped structures acquire distinguishing structural features.Hinges retain extended loop conformation in the domain-swapped form and are always in proximity with the other subunit. Considering extended conformation and proximity to the other protomer, hinge residues forms very few or no intramolecular interactions but shows high degrees of intermolecular interactions. This characteristic of hinge residues is very crucial in deciding precise boundaries of hinge region, when structural coordinates are known.The reliable prediction of which genes, and which regions of the genes, might be engaged in domain swapping could be useful for the design of bioengineering experiments and also to recognise potential target genes in various diseases.High quality domain-swapped dataset was obtained from 3DSwap+ database (manuscript under revision). In this updated version of the database, which is an automated and manually curated one, 2057 PDB entries of 3D-domain swapping protein entries are present with their sequence, structural and functional features.For the creation of negative dataset, Best Representative Profile (BRP) approach was used, which assigns a best representative sequence (BRS) and also a BRP to a Pfam protein family . All theProteinOrtho version 5.06, an orthology detection tool  was usedA total of 439 sequence features was used for training the model. Out of 439 features, 39 were physicochemical features of amino acids extracted from AAINDEX database  and 400 For the generation of the prediction model, 2000 sequences from 3DSwap+ were used as positive dataset and 2000 sequences created by BRP approach were used as negative dataset. A Perl script was used to extract the numerical values based on AAINDEX of all the features of amino acid residues. All the numerical values of positive and negative sequences were saved in two comma separated values (csv) files. These two files were given as input to RF and SVM classifiers to generate models for the purpose of classification and a confusion matrix of 2*2 with True Positive (TP), True negative (TN), False Positive (FP) and False Negative (FN) values for training dataset was also generated . The conk = 5). The original dataset is randomly divided into five derived datasets. Of these five datasets, a single one is retained as validation data for testing the classifier and remaining (5–1) derived datasets are used for training. The process is repeated five times (folds) with each of the derived datasets used only once as the validation data. These results from five-fold cross-validation datasets were averaged to obtain a final single estimation of statistical parameters.Evaluation of the prediction results was performed on a five-fold cross-validation method for the testing dataset. Cross-validation is a statistical technique for estimating the performance of a machine learning based prediction model. Fivefold cross-validation refers to an implementation of k-cross validation method . These measurements are expressed in terms of the fraction of true positives (TP), false negatives (FN), true negatives (TN) and false positives (FP).Sensitivity. Sensitivity refers to the percentage of sequences correctly predicted as “swapping” by the models.Specificity. Specificity refers to the percentage of sequences correctly predicted by the models as “non- swapping”.Matthew’s correlation coefficient (MCC). The MCC provides a combined measure of sensitivity and specificity. MCC ranges from –1 to 1. A value of MCC = 1 indicates the best correlation or possible prediction while MCC = -1 indicates the worst possible prediction or anti- correlation. Finally, MCC = 0 would be expected for a random prediction scheme.Accuracy. Accuracy refers to the percentage of correct prediction out of the total number of predictions.The RF and SVM models were applied to 137 reviewed, aggregation-related protein sequences from human proteome. These proteins are associated with different diseases such as amyloid forming proteins. The positively predicted 3D-domain swapping sequences were searched against PDB for their structural homologous at an E-value of 0.001.Mycobacterium tuberculosis (Mtb), Homo sapiens and Bacillus thuriengenesis (Bth) and also on few plant genomes as Solanum tuberosum (Stu), Ocimum tenuiflorum (Ote), Medicago sativa (Msa) and Arabidopsis thaliana (Ath). All the protein sequences of these genomes were downloaded from UNIPROT.Prediction models were applied on different genomes like All the 20247 reviewed entries of the human genome were downloaded from UNIPROT to predict 3D-domain swapping at the complete proteome level . For theDetailed analysis of these 3D-domain swapped predicted protein sequences  from humEspritz–an efficient online server for detection of protein disorder  was applHinge region prediction has been performed in the 3D-domain swapped predicted sequences by using a novel algorithm based on important features. The distribution of hinge region and swapped domain lengths were analyzed for the known examples of 3D-domain-swapped protein structures. On the basis of analysis done on the known examples of 3D-domain swapped molecules of 3DSwap+ database (~2000 entries), a minimum hinge size of three residues and maximum of seven residues were found. Six physicochemical features form hinge region sequences were employed in this algorithm. These features are (1) Propensity of amino acids in the hinges , (2) AveThe normalization of the score was done by following formula:Where,N = Normalized score of each residueS = Cumulative score of a residue in the sequenceX = Maximum score in the sequenceY = Maximum score of any residue in the given sequenceThe coverage of the tool was calculated using following formula:Where,C = CoverageRh = Number of proteins in, which whole or part of identified hinge region overlaps with reference hingeW = Number of proteins in, which, identified hinge and reference hinge are totally differentS1 FigExamples of challenging cases for hinge prediction, experimentally identified hinges are marked in blue color , B and C: Intertwined dimmers (PDB: 1A64), D: Missing regions (PDB: 2ZEJ), E: Very small hinges (PDB: 5CRO) and F: Elaborate interface (2PJW))(TIF)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(TXT)Click here for additional data file.S3 Table(TXT)Click here for additional data file.S4 Table(TXT)Click here for additional data file.


After Alzheimer, Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Alpha synuclein (SNCA) is deemed as a major component of Lewy bodies, a neuropathological feature of PD. Five point mutations in SNCA have been reported so far, responsible for autosomal dominant PD. This study aims to decipher evolutionary and structural insights of SNCA by revealing its sequence and structural evolutionary patterns among sarcopterygians and its paralogous counterparts (SNCB and SNCG). Rate analysis detected strong purifying selection on entire synuclein family. Structural dynamics divulges that during the course of sarcopterygian evolutionary history, the region encompassed 32 to 58 of N-terminal domain of SNCA has acquired its critical functional significance through the epistatic influence of the lineage specific substitutions. In sum, these findings provide an evidence that the region from 32 to 58 of N-terminal lipid binding alpha helix domain of SNCA is the most critical region, not only from the evolutionary perspective but also for the stability and the proper conformation of the protein as well as crucial for the disease pathogenesis, harboring critical interaction sites. PD is the second most common neurodegenerative disorder after Alzheimer which affects 1–2% of the population above age 65 and 4–5% above age 85SNCA is a 14.5 kDa, 140 a.a protein encoded by 5 exons with total transcript length of 3041 bps maps on 4q21.3-q22. The other members of synuclein family are SNCB and SNCG mapped to human chromosome 5q35 and 10q23.2-q23.3 respectively108813141514“critical region” of SNCA from evolutionary, functional and disease pathogenesis perspective.Due to the significant role of SNCA in FPD and other neurodegenerative disorders, this study was premeditated to decipher the molecular evolution of SNCA, which infers the phylogenetic history of synuclein family with the help of its putative orthologs and paralogs. Analysis revealed the sarcopterygian specific origin of SNCA which suggested its lineage specific functional role. On account of this interest, a comparative sequence and structural analysis was performed to estimate the selection and functional constraints on SNCA. Evolutionary rate difference was coupled with structural information to infer potential functional changes and the impact of lineage specific substitutions. In addition variations in domain topologies were explored by comparative analysis of known functional domains of SNCA protein. In light of the findings, it was hypothesized that the region from 32 to 58 of N-terminal lipid binding domain is the most Putative paralogs of human SNCA were determined by Ensembl Genome BrowserHomo sapiens (Human), Pan troglodytes (Chimpanzee), Mus musculus (Mouse), Rattus norvegicus (Rat), Gallus gallus (Chicken), Canis familiaris (Dog), Equus caballus (Horse), Loxodonta Africana (Elephant), Dasypus novemcinctus (Armadillo), Anolis carolinensis (Lizard), Pelodiscus sinensis (Chinese softshell turtle), Xenopustropicalis (Frog), Latimeria chalumnae (Coelacanth), Danio rerio (Zebrafish), Takifugu rubripes (Fugu), Tetraodon nigroviridis (Tetraodon), Gasterosteus aculeatus (Stickleback), Oryzias latipes (Medaka).Species that were selected includes The phylogenetic tree of SNCA family was reconstructed by using the neighbor-joining (NJ) method19Ancestral SNCA sequences were inferred by using ML method and WAG model of amino acid substitution. To investigate selection constraint within hominoids , non-hominoids , non-primate placental mammals  and non-mammalian tetrapods , z-test was implemented with MEGAa/Ks <1 were also classified on the basis of their physicochemical properties i.e. charge, volume, polarity into neutral or radical28Domains, motifs and sub-motifs have been assigned to human SNCA as described previously25For the structural analysis with evolutionary and functional perspective, NMR structure of human SNCA (1XQ8) was attained from Protein Data Bank (PDB)To conduct the interaction study, Cluspro protein-protein docking serverEvolutionary relationship between SNCA and its putative paralogs was estimated by NJ and ML methods . Phylogea/dN) and synonymous substitution rates (Ks/dS) were estimated for each group. And then z-test was applied to check selection constraint on the above mentioned groups.In order to estimate the evolutionary rate differences of SNCA gene among various clades of sarcopterygians, the orthologs of SNCA from representative members of hominoids , non-hominoids , non-primate placental mammals  and non-mammalian tetrapods  were obtained. Non synonymous (Ka-Ks (dN-dS) difference for hominoids was found as −2.241 (P = 0.014), non-hominoids was −4.716 (P = 0), non-primate placental mammals was −6.1777 (P = 0) and non-mammalian tetrapods was −7.085 (P = 0)  suggests negative selection, i.e. non silent substitutions have been purged by natural selection, whereas the inverse scenario (Ka > Ks) implies positive selection i.e. advantageous mutations have accumulated during the course of evolution. However the evidence for positive or negative selection requires the value to be significantly different from each other41The K= 0) see . In genea-Ks (dN-dS) difference for SNCB was identified as −1.661(P = 0.05) for hominoids, −4.708(P = 0) for non-hominoids primates, −5.212(P = 0) for non-primate placental mammals and −2.992(P = 0.002) for non-mammalian tetrapods (see a-Ks (dN-dS) difference for SNCG was identified as −1.658(P = 0.05) for hominoids, −4.064(P = 0) for non-hominoid primates, −5.485(P = 0) for non-primate placental mammals, −6.306(P = 0) non-mammalian tetrapods and −6.341(P = 0) for fishes   see . This da2 lipid binding alpha helix domain (1–60), Non-amyloid β component (NAC) domain (61–95) and C-terminal acidic domain (96–140)  as well as the paralogous copies in human (SNCB and SNCG). This annotation revealed the distinctive architecture of SNCA gene which is comprised of N-terminal A(96–140) .N-terminal lipid binding domain consists of 5 KXKEGV imperfect repeatsNAC domain forms the amyloidogenic core of SNCAC-terminal acidic domain harbors copper binding motif containing DPDNEA(119–124) consensus sequenceLineage specific substitutions that have occurred during evolution among analyzed sarcopterygians have been mapped on human SNCA with the help of ancestor reconstruction technique. It classified that nine substitutions have occurred at the root of mammalian ancestor. While two substitutions occurred at the root of non-primate placental mammal’s lineage and one occurred specifically to the catarrhini’s (hominoids and old world monkeys) lineage  Table 1Table 1. 2 alpha helix domain but for NAC domain as well. With the help of electron and X-ray diffraction techniques it has been reported that normal SNCA assembles through its N-terminal region which again highlights the significant role of this domainTo further inspect how purifying selection is performing its role in defining the spatial constraints on ancestral SNCA proteins at structural level, a comparative structural study was conducted. NMR structure of human SNCA (1XQ8) was taken as a reference and compared with modeled ancestral proteins . StructuIntriguingly, all human specific mutations involved in FPD pathogenesis reside in this crucial region which signifies that any change in this region will be deleterious because of the strong selection and functional constraints imposed on it. Superimposed mutant models with 1XQ8 identified major shifts toward lipid binding domain in A30P and H50Q, whereas major change was observed in lipid binding and NAC domains in case of E46K and A53T. Only G51D showed altered NAC region only see . All fivFurther to investigate the structural differences among the human paralogs of SNCA, a comparative structural analysis was also conducted. As the NMR structures of human SNCB and SNCG have not been reported so far, their structures were modelled by taking NMR structure of human SNCA (1XQ8) as reference and the structural deviations were assessed see . It appeIn order to investigate further the importance of this critical region, its functional significance was then deciphered with the help of interaction study. For this purpose, synphilin-1 (SNCAIP) was considered, as domain annotation of synphilin-1 revealed that it is 919 a.a (3745 bp) protein encoded by 10 exonsIn order to explore the role of critical region in interaction, docking analysis was conducted. Interactions have been identified between sarcopterygian ancestral, mammalian specific and non- primate placental mammal’s specific SNCA proteins, which revealed that interaction between SNCA and coiled-coil domain of SNCAIP has evolved with the passage of time i.e. lineage specific interactions have emerged during sarcopterygian evolutionary history . InteracInteraction analysis between mutant models of human specific SNCA with SNCAIP revealed altered interaction patterns while some of the wild type interactions were retained too which signifies that SNCA and coiled-coil domain of SNCAIP not only interact in normal individuals but also in the FPD patients, however the pattern of interactions was found altered. Docked complexes of A30P-SNCAIP and E46K-SNCAIP revealed that the interactions shifted entirely to the NAC domain whereas H50Q-SNCAIP and G51D-SNCAIP complexes showed altered interactions involving N-terminal and NAC domains. Only interactions in A53T-SNCAIP were confined entirely to N-terminal domain but the pattern was found altered. It can be assumed that the interactions altered due to differential interaction pattern of SNCA and SNCAIP thus affecting their binding affinities which in turn influence SNCA aggregation .Advent of high throughput annotation of genes has enabled bioinformatics analysis of genes of interest to provide important insight into their evolutionary link with particular phenotypic trait and association with human diseaseThe ML and NJ gene phylogenies of synuclein family revealed that SNCA is a sarcopterygian specific gene and thus pinpoints its functional specificity to this class. The three putative paralogs of this family form the (SNCG) topology indicating that SNCA and SNCB are closely related duplicate genes. Both of these paralogs share 63% identity which is also depicted in their cellular localization and functiona (dN) and Ks (dS) values were calculated within different phylogenetic groups of sarcopterygians. Estimation of statistical significance of difference between average Ka and Ks (z-test) within each phylogenetic groups of sarcopterygians have shown signature of strong negative selection on SNCA and its putative paralogous copies (SNCB & SNCG)  specifically in sarcopterygian lineage. For this purpose the average KNCG) see  which co protein a,b.“critical region” of SNCA by the phenomena called as “epistasis”. The interaction study conducted between SNCA and coiled-coil domain of SNCAIP revealed that the critical region harbors crucial interaction sites which enlightened the vital role of this region in normal cellular as well as disease processes  revealed significant structural deviations encompassing region 32 to 58 of N-terminal lipid binding domain of SNCA, despite of its high sequence conservation . The desrocesses . The funAlthough the normal cellular function and the metabolic pathways in which SNCA is involved are quite ambiguous, it has been reported that oxidative stress and proteasome inhibition are implicated in PD and other neurodegenerative disorders49Keeping in view the indispensable role of SNCA in the neurodegenerative processes, it is concluded that there are selective forces at work in sarcopterygians regulating the molecular and cellular mechanisms of SNCA. If this is the case, then fine tuning of these mechanisms through subtle changes in protein activity might be one of the contributing factors in bringing vital evolutionary changes to match the different environmental and ecological needs. The result of present study provides evidence that during the course of evolution, the region encompassed 32 to 58 of N-terminal lipid binding domain has acquired its critical significance in normal cellular function of SNCA and disease pathogenesis. The epistatic influence of the lineage specific substitutions might have led to the structural remodeling and functional innovation of SNCA. i.e., any mutation in this critical region is deleterious. These findings pave the way to investigate further the vital role of identified critical region of SNCA in different interaction studies and also with drug discovery perspective to target this region for the treatment of FPD.How to cite this article: Siddiqui, I. J. et al. The Parkinson Disease gene SNCA: Evolutionary and structural insights with pathological implication. Sci. Rep. 6, 24475; doi: 10.1038/srep24475 (2016).


PtdIns is synthesized from CDP-diacylglycerol and cytoplasmic inositol by PtdIns synthase (PIS) and is phosphorylated sequentially on its inositol ring to produce PtdIns4P and PtdInsP2 by PtdIns 4-kinase (PI4K) and PtdIns phosphate-kinase (PIPK), respectively  into the second messengers diacylglycerol and inositol-1,4,5-trisphosphate   is involved not only in the structural composition of eukaryotic cellular membranes but also in the regulation of a wide variety of physiological processes influencing growth and development  in A. thaliana, although AtPLC8 and AtPLC9 with amino acid substitutions in the Y domain do not group into the clade containing other PLCs during phylogenetic analysis P2-hydrolyzing activity, with an insertion at its N-terminal EF hand P and PtdIns(5)P, respectively, to produce PtdInsP2 P2 similar to yeast Fab1  shows similarity to animal PIPKs, whereas the domain structure of subfamily B members (AtPIP5K1-AtPIP5K9) includes a long N-terminal extension containing a repeat of the membrane occupation and recognition nexus (MORN) motif (Mueller-Roeber and Pical, A. thaliana and P. patens PIPKs (Mikami et al., A. thaliana contain only the B subfamily and a small number of genes for PIPK orthologs exist in C. reinhardtii (single copy; Awasthi et al., P. patens (two copies; Saavedra et al., The genome of E. siliculosus, indicating the presence of the MORN repeats is restricted into the green lineage, although PIPKs without the MORN motifs are conserved in A. thaliana, as mentioned above. Because the common origin of the red and green algae can be ascribed to a single symbiosis (Keeling, As shown in Figure Keeling, , there aComparative genomics provide an evolutionary insight into the taxonomic origin of enzymes and their isoforms, which is generally drawn based on the presence and number of gene family members. Here, it is demonstrated that analysis of domain structure can allow novel evolutionary conclusions. Because domain structure is involved in the regulation of the active state of each protein, comparison of plant genomes with a focus on the structure of and changes in protein domains could open new scenarios regarding the origin and evolution of signaling networks that regulate development and stress responses in plants.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies. Abs are secreted glycoproteins of ~150 kDa that represent the soluble form of the antigen receptor of B cells . The maji.e., “cytokine storm”, caused by massive activation of lymphocytes by Fc/FcR crosslinking) [A key limiting factor in the application of mAbs as bio-drugs is their pharmacokinetic properties . A fine linking) ,9, one slinking) . On the linking) . This prlinking) . Howeverlinking) .There has been great technological advancement in the field of development of new Ab-fragment based technologies, including generation of recombinant Fab fragments and nanobodies, the latter composed of a single functional variable domain originating from a parent mAb . While tThe Ab molecule is a tetramer composed of two identical copies of a glycosylated heterodimer that contains a heavy (H) and a light (L) chain covalently linked via disulfide bond. The H-L heterodimers associate in turn through their H chains by additional disulfide bonds . The Ig The Ig domain is also found in many other proteins besides Abs. A protein domain is classified as Ig or Ig-related when its structure, size and amino acid sequence are similar to the β-barrel found in Abs. All the Ig containing proteins constitute an evolutionary related group of proteins denominated the Ig-superfamily. Ig-superfamily members include surface proteins in cells of the immune and nervous system that regulate processes like cellular recognition and tissue adhesion. Interestingly, there are no residues conserved with 100% identity within the Ig domain in the Ig superfamily, not even the cysteines involved in the intra-chain disulfide bond, which are substituted by hydrophobic residues in some Ig-related proteins like CD2, CD4 and LFA3 . Thus, tThe quaternary structure of the Ab molecule is stabilized by the disulfide bonds found along the H and L chains . AdditioProteins in solution, and by extension the Ig domains in Abs and Ab fragments, are in equilibrium between their folded (native) and unfolded states . While tGiven the interest in developing Ab fragments smaller than a Fab, stabilization of Ig folding in Fv fragments has been pursued by different approaches. The design of a covalent disulfide bond between VH and VL domains of the Fv (dsFv) was one of the first strategies employed to reinforce the V interface ,29. AnotIn spite of the advantages of the multimeric Ab fragments described above, there is a strong interest in the field of Ab-based therapeutics to develop small monomeric Ab fragments consisting of a single V domain –45. The Interest was regained after finding that in camelids , and in sharks, part of the their humoral immune response includes Ab molecules that inherently lack light chains ,45,48. TMoving away from the hydrophobic VH/VL interface, a source of instability for the Ig folding in nanobodies is the CDR loops ,51. ThesIt seems as if natural evolution of VHH and V-NAR Ig domains is delivering structural solutions for the main limitations that nanobodies present as pharmaceutical drugs (Ig folding stability and Ag affinity). Strong efforts have been directed to either develop nanobodies based on these domains or to modify regular VH domains in an analogous fashion. Libraries of camelid nanobodies have been screened  in search of high affinity antigenic specificities against multiple targets such enzymes, haptens, viruses, toxins and venoms, cell surface tumor markers, and tissue markers for immune-imaging . RegardiOnce unfolding has taken place and the Ig structure is denatured, the tendency to aggregate depends on the solubility of the denatured Ig domains . When unIn order to prevent Ig aggregation, significant efforts have been directed to identifying structural determinants in different Ig domains that imprint high solubility in the non-native state. In principle, this knowledge could aid in engineering “super-stable” Ig domains that would remain soluble when exposed to a changing environment and tend to spontaneously refold back into native conformation. Comparison of the sequences of human VH and camelid VHH domains has allowed the identification of residues that in camelids are responsible for the low aggregation (high solubility) of unfolded VHH domains. The so-called VHH tetrad consists of 4 residues found to be key for high solubility in camelid VHH . These aUnder different kinds of environmental stress, like low temperature or high protein concentration, Abs and their derived fragments may aggregate . Ig domai.e., glycine, alanine, lysine, proline, taurine), methylamines ), polyalcohols and sugars , and urea. Osmolytes accumulate inside the cell to counteract osmotic stress [i.e., arginine, proline, TMAO) because they increase protein solubility and maintenance of protein native state, inhibiting protein aggregation  have an opposite effect, solubilizing proteins to a denatured state. The protective vs. denaturing character of osmolytes relies on their solvophobic/solvophylic interactions with the peptide backbone of proteins [Natural osmolytes are small organic molecules including certain amino acids and derivates  or high  temperatures, high concentration of salts , exposure to urea , and desiccation  . Interesproducts ,86. In oproducts . Osmolytproducts –90. Argiproducts , while gproducts ,93. As dproducts  (Figure products . InteresAn increased understanding of the mechanisms by which the Ig domain folds and retains its native/functional conformation will aid continued efforts to generate Ab-based therapeutics and diagnostics. In this review we have discussed the most recent findings regarding these questions that allow the development of different approaches to achieve stable Ig domains compatible with the manufacturing and commercialization of Ab fragment-based biotechnologies. Still better understanding of the Ig folding, regarding its stability and solubility in relation to changing environmental conditions is required to achieve the ultimate goal, obtaining a universal Ig scaffold that can function as a stable building block for different types of Ab fragments.