Multicellular organisms potentially show a large degree of diversity in reproductive strategies, producing offspring with varying sizes and compositions compared to their unicellular ancestors. In reality, only a few of these reproductive strategies are prevalent. To understand why this could be the case, we develop a stage-structured population model to probe the evolutionary growth advantages of reproductive strategies in incipient multicellular organisms. The performance of reproductive strategies is evaluated by the growth rates of the corresponding populations. We identify the optimal reproductive strategy, leading to the largest growth rate for a population. Considering the effects of organism size and cellular interaction, we found that distinct reproductive strategies could perform uniquely or equally well under different conditions. If a single reproductive strategy is optimal, it is binary splitting, dividing into two parts. Our results show that organism size and cellular interaction can play crucial roles in shaping reproductive strategies in nascent multicellularity. Our model sheds light on understanding the mechanism driving the evolution of reproductive strategies in incipient multicellularity. Beyond multicellularity, our results imply that a crucial factor in the evolution of unicellular species’ reproductive strategies is organism size. With The origin and the evolution of reproductive strategies are not well understood. Only a few reproductive strategies have been considered in previous work. The fragmentation mode of producing many-celled propagules has been investigated, in order to understand cell death in yeast  or to unOrganism size confers various advantages to organisms ,23, suchSaccharomyces cerevisiae [Volvox, along with the germ–soma differentiation [Previous studies have shown that cellular interactions can change reproductive modes ,23,28. Frevisiae . Phenotyrevisiae ,35. In tntiation , 1–20 gentiation .Thus, the size and composition could affect growth in phenotypically heterogeneous multicellular organisms. We develop a theoretical model to address the evolution of reproductive strategies considering the effects of organism size and thresholds for the number of different cell types. The size effects could increase or decrease organism growth, with the organism growing faster when the cell number of a particular phenotype reaches a given threshold. Organisms in a population share one common reproductive strategy while populations differ in reproductive strategies. Reproductive strategies thus compete with each other via population growth rates. The optimal reproductive strategy maximizes the population growth rate. We found that reproductive strategies can coexist or dominate others under different conditions. The uniquely optimal reproductive strategy always produces two offspring units.. 2a). The organisms in each population have a unique reproductive strategy. For example, a population with maturity size N = 3 must have the reproductive strategy either 1 + 1 + 1 or 2 + 1. In a population with 2 + 1, mature organisms with three cells produce a single-celled newborn organism and a two-celled newborn organism. The reproductive strategy determines the organism size at which an organism is born and at which size it is mature and reproduces. For the reproductive strategy n1 + n2 + · · · + nM, newborn organisms have cell number ni  and maturity size n1 ≥ n2 ≥ · · · ≥ nM. We consider organisms consisting of two cell types: type A and type B. Newborn organisms may differ in their size and composition in a population. For example, a population with reproductive strategy 2 + 1 can have five types of newborn organisms: , , ,  and , where  shows the number of type A cells and type B cells in an organism (d). Each organism grows incrementally by one cell at a time. During each increment, a cell is selected to divide, and two daughter cells are produced. Each daughter cell can switch to another phenotype independently with a cell-type switching probability, which is typically m = 0.01 in our model . After reaching their maturity size N, organisms reproduce via random fragmentation in terms of organism composition. The probabilities of forming different newborn organisms are calculated in electronic supplementary material, appendix S1. The newborn organism follows the same life cycle, growing from newborn to mature (see a).We consider multiple populations in which organisms grow and fragment into smaller pieces a. The ororganism d. Each oture see a.Figureλ, depending on the number of offspring and the growth time of organisms [tn is the time for organisms growing from size n to (n + 1). The growth time depends on the cell size at the newborn stage and the cell size at the maturity stage. The time tn is determined by the organism size and composition astsn and tgn are the size component and the threshold component of tn. Next, we discuss how we model tsn and tgn.We assume that organisms in populations grow independently without density dependence. Thus, populations follow exponential growth . The poprganisms ,38, can tsn depends only on the cell number n = nA + nB of an organism during growth, but not on nA or nB individually. Under the neutral condition b). Without loss of generality, we choose γ = 1 and treat tsn around the neutral condition (a). We call n = 1, …, N. By perturbing all normalized cell increment components χn for n = 1, …, 7, we can capture all possible effects of size. For χn = 1, we recover the neutral condition.The size component ism size . Thus, osn0) see a. We caltgn is determined by the number of the cell type of interest. Without loss of generality, we choose the type B. We assume the type B providing a benefit to the organism while bearing an individual cost. An organism grows faster if the number of its type B cells meets a given threshold k (c). There are many methods to construct such a compositional threshold effect. Here, we choose a volunteer dilemma game [n cells with nA type A cells and nB type B cells. When nB meets a contribution threshold k, each cell of the organism gets a benefit b from an increased organism growth. Each B cell pays an individual cost c and A cells pay no costs (d):pA and pB are the division probabilities for A cells and B cells, respectively, and w is the intensity of selection [tgn is the inverse of average fitness of the organism:k.The threshold component eshold k c. There mma game . Consideno costs d:2.2PA. If the organisms of a population are going through a perturbed state at size n, i.e. n1 + n2 + · · · + nM) can deviate from the one under the neutral condition. Since the population growth rate is inversely proportional to growth time, a perturbation is either advantageous  or disadvantageous  for population growth. A reproductive strategy is referred to as being promoted (suppressed) when its population growth rate is greater  than the neutral growth rate 1. A single advantageous perturbation (χn < 1) promotes the reproductive strategy of any population with organisms going through the state n of the perturbation, i.e. the strategies are satisfying nM ≤ n ≤ N (b). The performance of reproductive strategies is unaffected when their populations’ organisms do not go through the size under perturbations, i.e. n < nM or n > N. A single adverse perturbation χn > 1 suppresses reproductive strategies that satisfy nM ≤ n ≤ N. Among these affected populations, we found that the reproductive strategy n + 1 is most affected by perturbations at size n. Since the population with reproductive strategy n + 1 contains n-celled newborn organisms, which mature at size n + 1, its growth time depends on χn. Therefore, under the condition of χn < 1 and χk = 1 , the reproductive strategy n + 1 is uniquely optimal. Under the condition of χn > 1, the reproductive strategy n + 1 is most suppressed (see b). Analogous to the reproductive strategy n + 1, the reproductive strategy n + 2 is the second most affected reproductive strategy. Similarly, for the rest of the reproductive strategies, their population composition determines whether the growth rates are affected or not. The growth rates then determine the performance of reproductive strategies.To focus on size effects only, we first assume no threshold effect,  1, …, 7 a. If the ≤ n ≤ N b. The pessed see b. Analogn = 1, …, 7, which forms a discrete size function with respect to n. As each χn is independently perturbed from the neutral condition, the discrete function can cover all possible shapes. We investigate the general features of size functions that promote a given reproductive strategy, but ignore the specific shape of the size functions. As expected, we observed that the populations of optimal reproductive strategies contain organisms that mostly go through sizes with smaller χn. This is illustrated in figure 2c and an analytical proof is given in electronic supplementary material, appendix S3, for reproductive strategies with N ≤ 3. We found that only the binary-splitting reproductive strategy (producing two offspring) can be uniquely optimal . Intuitively, this is because the fastest-growing newborn organisms in a population with a multiple-splitting reproductive strategy can also be found in another population with a binary-splitting reproductive strategy. For example, the population growth rate of 2 + 1 + 1 cannot be greater than that of 1 + 1 and 2 + 2 at the same time. Additionally, 1 + 1 is the most frequently observed reproductive strategy in binary-splitting reproductive strategies (figure 2d) because 1 + 1 is the only reproductive strategy that depends on a single cell increment component χ1. Therefore, for a randomly chosen χn , 1 + 1 has a higher probability of being optimal compared to other strategies. Generally, reproductive strategies have lower chances to be optimal when binary splitting makes organisms go through many cell increment stages.We can analyse all possible size effects by combining single perturbations for . 3.2χn = 1, such that b − c = 0, cells of type B would never lead to any growth advantage; thus we focus on b − c > 0. With a threshold at size k, newborn organisms of a population with nB ≥ k have larger payoffs and thus have shorter growth time; see equations (d). With the contribution threshold k = 2,  grows fastest as it has two B cells.  is the second-fastest-growing newborn organism as it most likely gains benefits by producing a second B cell during growth. ,  and  grow relatively slowly because they are less likely to produce at least two B cells during growth. For convenience, we refer to newborn organisms in a population as ‘beneficial’ if nB ≥ k and ‘intermediate beneficial’ if nA = 0, nB < k and N > k, where N is maturity size. All other newborn organisms are unlikely to reap the benefits of cooperation before maturity. The growth rate of a population depends primarily on its beneficial newborn organisms and secondly on its intermediate beneficial newborn organisms. Moreover, among all beneficial newborn organisms, the one with exactly k type B cells has the largest payoff. Thus, they have growing advantages under no or low cell-type switching probability. For a low cell-type switching probability, e.g. m = 0.01, homogeneous newborn organisms are more abundant than heterogeneous ones. In the long run, we expect that populations mostly contain homogeneous newborn organisms.To investigate threshold effects exclusively, we assume the size effect to be neutral: quations  and (2.4quations d. With ta). The optimal reproductive strategies can be classified into three categories: multiple optima, symmetric binary splitting (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) and asymmetric binary splitting with a k-celled newborn organism (N − k) + k. When k = 1, multiple reproductive strategies are optimal at the same time (see a–c). Since every population contains beneficial newborn organisms, the performances of different reproductive strategies are similar. As k increases, the symmetric binary-splitting reproductive strategies (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) are optimal for 1 < k ≤ (1/2)N . Newborn organisms with size equal to or greater than k have growth advantages; thus, intuitively (N/2) + (N/2) and k + (k + 1) should have the same performance in population growth. However, we found that only (N/2) + (N/2) (or ((N + 1)/2) + ((N − 1)/2)) is optimal. The intrinsic composition of the population and the effects of cell-type switching probability m = 0.01 determine the results. To understand the growth advantages of the symmetric binary-splitting reproductive strategies with the maximal maturity size, we take 4 + 4 and 3 + 3 at k = 3 as an example. For k = 3, the population of 4 + 4 contains the beneficial newborn organisms  and . The population of 3 + 3 only contains beneficial newborn organisms . When a cell-type switching event happens during growth,  produces a beneficial newborn organism , while  reproduces a non-beneficial newborn organism . Populations with larger maturity sizes are less affected by the cell-type switching probability as they contain multiple types of beneficial newborn organisms. Finally, when N − k) + k becomes optimal (see a). When N − k) + k rather than other reproductive strategies such as N − k − 1) + k + 1. Because of N − k < k, organisms with N − k cells can only form intermediate beneficial newborn organisms—and only when they are pure B cells. Larger intermediate beneficial newborns grow faster than smaller ones. We take 3 + 1 + 1 and 3 + 2 for k = 3 as an example. 3 + 1 + 1 has the intermediate beneficial newborn organism  and 3 + 2 has the intermediate beneficial newborn organism . During organism growth,  undergoes two cell increment stages with longer time N see a,b. Newbimal see a. When kquations  and 2.2a. The opk, resulting from reducing the number of beneficial and intermediate beneficial newborn organisms. Especially when k ≥ N, no reproductive strategies will obtain the benefits of cooperation, and their populations grow slower due to the associated costs . Increasing maturity size N increases population growth rates of the optimal reproductive strategies because the number of beneficial or intermediate beneficial newborn organisms increases. As expected, population growth rates also increase with the benefit to cost ratio (see b–e).Population growth rates decrease with increasing osts see a,b. Incratio see b–e.m on the reproductive strategy for N = 8. We found that it is robust for the conclusion: the optimal reproductive strategy is binary splitting with maximum maturity size. At high m and high k, the asymmetric binary-splitting strategy 7 + 1 invades other asymmetric binary-splitting ones . Since 7 + 1 can keep the largest number of B cells in a newborn organism, this organism could gain growth advantages for high cell-type switching probability. A higher cell-type switching probability increases the heterogeneity of organisms, especially for large organisms. This leads to fewer B cells in an organism—thus, the population growth rate generally decreases with increasing m; see electronic supplementary material, figure S2b. For some reproductive strategies, a change in the cell-type switching probability leads to complex effects on population growth . This is the outcome of several factors: the cell-type switching probability m, the contribution threshold k and the structure of the population. We take the reproductive strategy 1 + 1 + 1 as an example to discuss the potential reason: when k = 2, the population has a lower growth rate when m = 0.5 because organisms cannot reach the threshold during their growth.Additionally, we investigated the effect of varying . 3.3ni + nj can be uniquely optimal, where ni and nj are positive integers, and ni + nj ≤ N . With the combined effects of size and threshold, we found new optimal binary-splitting reproductive strategies that are neither optimal in the effects of single perturbation only nor for thresholds only, including 2 + 2, 3 + 2, 4 + 2, 5 + 2, 3 + 3 and 4 + 3. Furthermore, under a beneficial size perturbation, we found n + 1  can be optimal both at small and large contribution threshold k . This is due to the fact that the threshold effects lead to a similar performance of reproductive strategies either at small k and at large k (b). Therefore, for combined size and threshold effects, the size effect has a larger impact on the performance of reproductive strategies . Consequently, the reproductive strategy n + 1 becomes optimal under an advantageous perturbation, where n = 1, …, 7. Newly emerged binary-splitting reproductive strategies have advantages for intermediate contribution thresholds k, suggesting that it is an outcome of the trade-off between the effect of size and threshold. For an adverse size perturbation, we found that the reproductive strategy n + 1 cannot be optimal (b), because the adverse size perturbation leads to poor performance of reproductive strategies that are influenced by the perturbation (b and 4d). The reproductive strategy 7 + 1 is an exception to this rule, as the threshold effect strongly influences it at k = 7. The optimal reproductive strategies observed are those that can obtain growth benefits from threshold effects and avoid the disadvantages from the adverse size effect. For example, 3 + 3 outcompetes 4 + 4 for k = 2 when a disadvantageous size perturbation occurs at n = 7. Both strategies can obtain growth advantages from threshold effects. However, adverse size perturbation decreases the population growth rate of 4 + 4, but it has no impact on 3 + 3. Thus the performance of reproductive strategies is the outcome of the trade-off between the effects of size and threshold. Our results suggest that all binary-splitting reproductive strategies can evolve under an appropriate choice of size effects (at a single size) and threshold effects.Finally, we investigate the optimal reproductive strategies under size and threshold effects combined. For simplicity, we only consider the size effects in the form of a single perturbation. All binary-splitting reproductive strategies + nj ≤ N a,b. Withld k see a,b. This large k b. Therefgies see a,c. Cons optimal b, becausurbation b and 4d.. 4Numerous reproductive strategies are conceivable for multicellular organisms, but only recently more attention has been paid to the evolution of such reproductive strategies ,20,41,42n + 1 resembles organisms passing through a unicellular phase, which is a striking feature for most multicellular organisms [n + 1 can evolve under size or threshold effects. This indicates an alternative way for evolving organisms with a single cell bottleneck. Our models also show the conditions where multiple reproductive strategies can evolve simultaneously. Each reproductive strategy can evolve when the size effects are the same for each cell size (χn are the same) or when the threshold is low . Many reproductive strategies have beneficial newborn organisms (nB ≥ k) when the threshold is low, leading to a large population growth rate. This result indicates that multiple reproductive strategies can coexist for an organism. Notably, the results resonate with the observation that one species can possess several reproductive strategies simultaneously in nature [The reproductive strategy rganisms . Previourganisms ,17. Inten nature ,15,45,46χn), we found the frequently observed optimal reproductive strategy 1 + 1. The finding suggests that 1 + 1 is the best reproductive strategy unless explicit advantages exist for a larger organism. The size effects considered in our model are functions of cell numbers rather than the task allocation of each cell. Previous work on the division of labour has instead focused on continuous functions [In our model, we can choose a flexible impact of size on organism growth. We investigated the size effects by first investigating the effects of single size perturbations. Although single size perturbations seem unrealistic for organism growth in nature, they provide a theoretically convenient approach towards understanding size effects. Size effects that are present by combing single size perturbations could have positive, negative or neutral effects on growth at each cell increment. The model assumption corresponds to studies concerning size effect on growth –34. The unctions ,48–50.We delineated the threshold effect of cellular interactions in a multiplayer volunteer game given the utility of game theory in depicting biological interactions ranging from social foraging to cancer development –58. We dm = 0.01. This focus assumed that cell-type switching is uncommon and mostly happens under environmental pressure in nature [nB ≥ k) ) newborn organisms, then homogeneous beneficial  newborn organisms dominate the population. Although heterogeneous beneficial newborn organisms grow fastest, they are not most abundant. First, heterogeneous beneficial newborn organisms do not produce themselves fast after reproduction. The reason is that A cells have higher payoffs; thus, they produce more A cells during the growth of heterogeneous beneficial newborn organisms. When the organisms reproduce, they are unlikely to produce more than one heterogeneous beneficial newborn organism. Second, although heterogeneous beneficial newborn organisms can be replenished from the reproduction of beneficial newborn organisms (via cell-type switching), the replenishment is slow as beneficial newborn organisms have lower growth advantages. Thus, it is also theoretically intriguing to investigate reproductive strategies under high cell-type switching probability. Such an investigation shows that the conclusion is robust across cell-type switching probability parameter space: the optimal reproductive strategy is the binary-splitting one with maximum maturity size . Although high cell-type switching probability mainly decreases the population growth rate, nevertheless it does not affect the relative performance of reproductive strategies. Within these binary-splitting strategies, the binary-splitting reproductive strategy producing the largest newborn invades others at high cell-type switching probability.We mainly investigated reproductive strategies with a small cell-type switching probability, n nature ,60. The Stanieria, Pleurocapsa and Dermocarpella [Our model investigated the competition of reproductive strategies via the growth rates of isolated populations. In many situations, this correlates with the ability to invade other populations—simply by outgrowing existing types. We can extend the model to a density-dependent population consisting of organisms with different reproductive strategies. Given the size and threshold effects, the invasive strategies would reflect the reproductive strategies observed in our model. Furthermore, our model considered the size effect via the number of cells in an organism, so the size function is discrete. In nature, some unicellular organisms grow by enlarging themselves rather than by cell divisions, such as carpella . Insteadructures ,62.


The study examined the development of Chinese as a second language learners’ formulaic knowledge through comparing the processing of Chinese idioms versus non-idiomatic formulaic sequences (FSs) by advanced-level learners (ALs), super-advanced learners (SLs), and native speakers (NSs). Using two phrase acceptability judgment tasks with and without think-aloud protocols, we collected data on participants’ processing accuracy, processing speed, and processing strategies of reading the two types of FSs. Four processing patterns emerged from the analyses of the datasets. First, learners’ processing accuracy and speed increased along with their proficiency. Second, learners’ idiom processing ability was generally lower than that of non-idiom processing ability, but they demonstrated an improving trend as their proficiency level increased. Third, learners’ use of processing strategies did not change much as proficiency rose and demonstrated a categorical difference from NSs. Fourth, all three groups exhibited poorer productive idiom knowledge than productive non-idiom knowledge. The overall findings denote that second language learners’ formulaic knowledge can evolve beyond the lexical plateau as learners move from the advanced to a higher proficiency level, but the productive idiom knowledge can be a long-term problem. The findings provide implications for measuring and teaching Chinese formulaic knowledge at the higher-than-advanced stage. In second language acquisition (SLA), the two primary goals are to identify the nature of learners’ linguistic knowledge and to provide accounts for how this knowledge transforms over time . SpecifiIn the last two decades, mounting evidence has suggested that overall second language (L2) proficiency can be improved by acquiring formulaic sequences (FSs), which are multiword expressions that frequently recur as a whole in language use . This clDespite the general acknowledgment of the importance of learning formulaic knowledge, little is known about how formulaic knowledge develops as learners’ proficiency increases. Some corpus-based studies e.g.,  focused In the FS literature, there is a severe lack of studies gauging the development of L2 formulaic knowledge. This scarcity of research may have to do with certain characteristics of FSs. We review these characteristics and the related empirical findings in the following sections.Formulaic sequences lack homogeneity. Different types of FSs vary greatly in their linguistic dimensions, such as idiomaticity, form fixedness, function, and linguistic register . For exaIn NSs’ FS processing, although researchers generally agree that FSs have a processing advantage over non-formulaic novel phrases, In L2 learners’ FS processing, studies focusing on different kinds of FSs have often reported different processing patterns. For example, by examining idiomatic FSs, Due to conflicting findings, researchers have recently begun to call attention to the necessity of empirically comparing different types of FSs in SLA-oriented studies to discern the sources of learners’ processing difficulties, and to better integrate findings about FS processing into FS acquisition . The mosChinese FSs share the universal characteristics that have been observed in FSs of other languages. They also lack homogeneity, and studies on the processing of different types of Chinese FSs by CSL learners have also reported conflicting results. For example, set-language  and 惯用语 Guan-yong-yu habitual-use-language , may be the thorniest type for L2 learners. Chinese idioms are often “composed in Classical Chinese and thus typically have a different grammatical structure from that of Modern Chinese” Do ALs, SLs, and NSs demonstrate different patterns in FS processing accuracy?(2)Do ALs, SLs, and NSs demonstrate different patterns in FS processing speed?(3)Do ALs, SLs, and NSs demonstrate different patterns in their FS processing strategies?HSK:HangyuShuipingKaoshi) volunteered to participate. Before the main test, the participants took an online character quiz designed and distributed by the APP Wenjuanxing questionnaire. On the quiz, the participants were asked to associate each character with its correct meaning on a multiple-choice task. The characters included (but were not limited to) all the characters that would appear in the testing materials. All characters were within the required vocabulary range of the HSK 6 guidelines, so presumably the participants would already know all the characters. Knowing all target characters is important because it can confirm that any observed processing problems are NOT due to the presence of unknown words in the FSs. Finally, we included 26 learners (mean age = 26) who achieved a score of 100% on the quiz on the main test. Another 13 NS participants were undergraduate and graduate students recruited from two universities in Beijing (mean age = 27). None of them were Chinese language or literature majors.The participants included 13 ALs, 13 SLs, and 13 NSs. Although a larger sample size is always desirable for lexical research to iron out individual variances, The 26 L2 participants came from four Chinese universities and were enrolled in three types of programs: (1) language degree programs designed for foreign students to learn Chinese for general and specific purposes; (2) discipline programs  that mostly enroll Chinese undergraduate students and some very advanced foreign students who are able to use Chinese in academic settings; and (3) preparatory programs designed to prepare advanced foreigners to enter into discipline-focused programs. The background information about the L2 participants is presented in t-tests confirmed that the two groups’ HSK scores were significantly different . Other than HSK scores, we also considered years of learning the language and current enrollment programs. n of learners in a discipline-focused program = 8, n of preparatory program learners = 4, n of language program learners = 1) would be considered more advanced than the bottom 13 scorers, who were mostly learners in language programs . Moreover, We further divided the 26 learners into two proficiency groups. Because the HSK test does not have official guidelines to differentiate advanced and higher-than-advanced levels of proficiency, we used learners’ HSK scores as the primary criterion to distinguish their proficiency levels. We grouped the 13 highest scorers together (mean score = 247.77) and the 13 lowest scorers together (mean score = 206.54). Independent not be read in a verbatim fashion.The two types of FSs used in this study were Chinese idioms and non-idiomatic FSs. Because we differentiated FSs by the domain of idiomaticity, we treated subtypes of non-idiomatic FSs  as one category. For both idioms and non-idioms, we also manipulated the sequence length by using three-character (3-C) sequences and four-character (4-C) sequences with matched whole-form frequency. The rationale is that because 4-C items are one character longer than 3-C items, if 3-C items are processed significantly faster than 4-C items of the same FS type, then it is very likely that the FSs will be visually recognized in a word-by-word fashion. If the length does not play a significant role in processing speed, then it is more likely that the FSs will The Contemporary Chinese Dictionary (6th edition). We tried to be as exhaustive as possible. This procedure yielded a total of 2,098 4-C idioms and 3,783 3-C idioms. The 4-C idioms are all 成语 Cheng-yu; the 3-C idioms include both 惯用语 Guan-yong-yu and semantically non-transparent, verb-complement structures . Second, we ran the two types of idioms in the Google 1-gram database. Given the raw token frequency, we further extracted the top-ranked 300 items in each type. Then, by consulting with two experienced Chinese language teachers, we selected 38 3-C and 38 4-C idioms that are more likely to be familiar to advanced learners and have comparable whole-form frequencies for the idiom pool. Finally, using a 5-point scale, 33 Chinese language teachers rated these idioms based on the likelihood that an advanced Chinese learner would know them. Finally, we included 24 3-C idioms and 24 4-C idioms that received an average score of 4 or higher  they are grammatical phrases commonly used in modern Chinese; (2) they have equal numbers of characters and similar constructions to the matched idioms; (3) they share at least one identical content character with the matched idioms; and (4) they have comparable total stroke numbers and whole-form frequency to the matched idioms. Independent samples t-tests (a = 0.0125) showed no significant differences in the 3-C idiom and 3-C non-idiom frequencies  or stroke numbers . The 4-C idiom and 4-C non-idiom frequencies were also not significantly different , nor were their stroke numbers . The Independent samples t-tests also confirmed that the 24 3-C idioms and 24 4-C idioms were matched in whole-form frequency , as were the 24 3-C non-idioms and 24 4-C non-idioms .Because we aimed to compare the processing of idioms and non-idiomatic FSs, we matched each idiom with non-idiomatic FSs through the following steps. We first changed one constituent word of the target idiom and waited to see if the new expression would be grammatical, and if the whole-form frequency and total stroke number were matched with the target idiom . If changing one character failed, we continued to change two characters . If changing two characters also failed, we used the fuzzy search function provided in the BCC corpus and looked for a potentially non-idiomatic expression that shared at least one content character with the target idiom . After several rounds of matching, we found that some of the chosen non-idioms were absent from the existing Chinese frequency corpora. To solve this problem, we adopted n = 24) and (2) matched 3-C non-idioms (n = 24) and (3) 4-C idioms (n = 24) and (4) matched 4-C non-idioms (n = 24). Another 96 ungrammatical phrases were made up as fillers. We divided the test stimuli into two counterbalanced blocks so that the participants would not read an idiom and its matched non-idiom in the same block.In sum, the test stimuli in  consisteWe programmed the speedy acceptability judgment task using Paradigm software to collect judgment accuracy data and response time data. Acceptability judgment task paradigms have been widely used in the SLA field to measure learners’ L2 competence e.g., . BecauseWe conducted the think-aloud phrase acceptability judgment task 1 week after each participant finished the speedy acceptability judgment task. This task allowed us to examine participants’ thought processes when they judged the phrases. The purpose was to infer what processing strategies were used by different groups of participants and for different types of FSs. As a versatile data collection tool, the think-aloud method has been broadly employed in L2 studies to gather data about learners’ thought processes to measure processing strategies , depth oIn previous SLA research, think-aloud protocols can be classified into two types—metalinguistic and non-metalinguistic—each tapping into different kinds of knowledge and having different techniques. The metalinguistic think-aloud protocols—which are carried out by asking participants to provide justifications for their performance—tap into metalinguistic knowledge. The non-metalinguistic think-aloud protocols—which are conducted by simply asking the participants to verbalize whatever is on their minds while performing a task—tap into general cognitive processes see . The disI just feel it’s correct” or “我听过这个 I’ve heard of this.” On the test, we used the same set of test stimuli and a different set of filler stimuli. The test stimuli were presented one by one on a computer screen controlled by a research assistant. Because L2 learners were also asked to verbalize in Chinese, they were informed that they need not worry about language errors, because the purpose of the task was to understand their thought process, rather than testing verbal proficiency ”“请判断这个短语在汉语里能不能说，请说出您是怎么知道的。就把您思考时在脑子里对自己说的话大声说出来，就好像您独自坐在一个房间里跟自己说话一样。不用在意说的句子语法对不对，完整不完整，因为我们只想了解您在做判断时的想法。 ”. When developing the coding book, we consulted one grammaticality judgment study and one idiom processing study. In We coded the participants’ think-aloud reports for processing strategies. In this study, “processing strategy” refers to what type of information a participant relied on to make a judgment. A participant could use his/her intuition and say, “n = 750) independently. The intercoder reliability reached 95.3% (Cohen’s Kappa = 0.829). The agreement was considered high enough for one assistant to code the remaining data alone.Two trained research assistants coded approximately 20% of the think-aloud data . To analyze processing speed, we transformed the raw response time data by log10 to reduce skewness . We excluded incorrect responses to target items, which led us to remove 1.9% of the NS data, 5.7% of the SL data, and 16.3% of the AL data. We further trimmed the extreme data by setting the cutoffs at three standard deviations from each participant’s mean response time. This procedure removed an additional 1.1% of the NS data, 0.7% of the SL data, and 0.6% of the AL data. To analyze processing strategies, we included all think-aloud data .To analyze processing accuracy, we included all judgment data. Because the selected testing materials were highly familiar to NS participants and most non-native participants, the dichotomous judgment data (processing accuracy) were supposed to be positively skewed  and the interaction of group × type  and type × length . The effect of type was marginally significant , while the effect of length  and the group × length interaction  were both non-significant.The dichotomous judgment data were then analyzed via a generalized linear mixed-effects model in R using the lmerTest package. When fitting the models, we entered the variables of group, length, and type as fixed effects. We added the participants and items as random effects, including the random intercepts of participants and items and the random slopes for all fixed effects. When dealing with the multicollinearity issue, we excluded the three-way interaction of group × type × length from the model because the two-way interactions and the planned p < 0.000), NSs and SLs (p < 0.000), and ALs and SLs (p < 0.001). NSs’ judgments were more accurate than those of the two groups of learners, and SLs’ judgments were more accurate than those of the ALs. The analyses for the effect of type × group indicate that idioms were judged significantly more accurately than non-idioms by NSs (p = 0.012), while idioms were judged significantly less accurately than non-idioms by ALs (p = 0.024). SLs’ judgments of idioms and non-idioms were only marginally different (p = 0.061). This finding suggests that NSs’ recognition of idioms was more successful than their recognition of non-idioms, implying that idioms were more deeply entrenched in the NSs’ lexicons and therefore unlikely to be recognized incorrectly. For CSL learners with a relatively low proficiency level, the results hint that they were better at judging semantically transparent or syntactically regular sequences than the irregular formulae. For learners with a higher proficiency level, their judgment ability of the irregular formulae developed and approached their judgment ability of the regular formulae. The analyses for the effect of length × group demonstrated no significant results for NSs (p = 0.623), SLs (p = 0.297), or ALs (p = 0.747), thereby suggesting that longer FSs were recognized as successfully as shorter FSs by all three groups of participants.We then conducted the planned pairwise comparisons. The analyses for the effect of group show that the judgment accuracy was significantly different between NSs and ALs , length , the interaction for group × type , group × length , and group × type × length . The effect of type  and the interaction for type × length  were non-significant.The log10-transformed response time data were then sent into a linear mixed-effects model with group, type of FSs, and length of FSs included as fixed effects. We added the participants and items as random effects, beginning with a maximal random effects structure encompassing random intercepts for participants and items, and random slopes for all fixed effects. The outcomes showed a clear effect for group (p < 0.000), NSs and SLs (p = 0.017), and ALs and SLs (p < 0.000). Further, NSs outperformed the two learner groups, and the SLs outperformed the ALs. The analyses for the effect of group × type revealed that idioms and non-idioms were processed at a significantly different speed by NSs (p = 0.002), with idioms being processed faster than non-idioms, and by ALs (p = 0.019), with idioms being processed more slowly than non-idioms, but the difference was non-significant for SLs (p = 0.268). This pattern indicates that learners with lower proficiency might spend more cognitive effort processing semantically or syntactically irregular formulae than regular ones. As proficiency approached the near-native level, the cognitive efforts spent on irregular forms decreased, thereby generating comparable processing speed for idioms and non-idioms. For NSs, the processing pattern was reversed, as idioms were processed more effortlessly than non-idioms, suggesting that idioms might be pre-listed in NSs’ mental lexicons. The analyses for the effect of group × length showed that the processing speed of 3-C versus 4-C FSs was significantly different for the NS group (p = 0.002), with 4-C FSs being processed faster than 3-C FSs. A significant length effect was also found for the SL group (p = 0.002) and the AL group (p = 0.002). However, both with 3-C FSs were processed more quickly than 4-C FSs. The findings of the length effect for SLs and ALs were explainable, as the longer sequences required a longer time to read. Notwithstanding, the finding for NSs was counterintuitive. We computed the planned pairwise comparison for length × type for the NS group. The length effect existed only in the processing of idioms, with 4-C idioms responding significantly faster than 3-C idioms (p = 0.011), but the difference between 3-C non-idioms and 4-C non-idioms was non-significant (p = 1.00).To explore the differences between the levels of each variable, we performed planned pairwise comparisons. The analyses for the effect of group showed that processing speed was significantly different between NSs and ALs  by group. The results are presented in RQ3: It is just an idiom.” Learners either relied on their feelings or confessed that “I have learned this but forgotten its meaning.” When using intuition with non-idioms, all three groups of participants provided a cliché type of answer, such as “A just means A” or “It is just what I often say” without further elaboration. That NSs could quickly recognize idioms suggests that idioms are a very salient FS type in NSs’ mental lexicons. In contrast, learners’ feelings and confessions imply that idioms might not have been fully acquired. The cliché reports hint that both NSs and learners’ non-idiomatic FS knowledge had become proceduralized.Intuition was used more frequently by NSs; 38.8% of idioms and 35.9% of non-idioms were processed with intuition by NSs. SLs used intuition for 22.3% of idioms and 28% of non-idioms. ALs used intuition for 25% of idioms and 19.4% of non-idioms. When using intuition with idioms, NSs often immediately identified the idioms, saying, “The example strategy was more frequently used for non-idioms than idioms by all three groups. NSs provided examples for 53.4% of non-idioms, SLs for 45.7% of non-idioms, and ALs for 40.5% of non-idioms. For idioms, an example strategy was used 31.4% of the time by NSs, 19.9% of the time by SLs, and 17.9% of the time by ALs. In using the example strategy, the participants either described a typical situation in which an FS often occurs or directly used the target FS to make a sentence. Being able to use an FS in a real situation is evidence of the possession of proceduralized knowledge . The preTarget idiom: 脱口而出 cast-mouth-and-out “blurt out”I ‘tuo-kou-er-chu’ and spit sputum.)”An NS’s report: “我脱口而出吐了一口痰. .”An AL’s report: “嗯，这个，看起来是对的。比如说，嗯，我、我、我好久都没有跟你说什么，终于 脱口而出. ; the other is to propose an interpretation for an unfamiliar FS (which is the case for some learners’ interpretations).In the latter case, learners are often based on the constituent word’s meaning and their logical thinking to make sense of the unknown FS, sometimes resulting in an erroneous interpretation. An example is shown below.Target idiom: 千方百计 thousand-method-hundred-plan “to make every possible effort to”Correct. Should…I think that the meaning should be ‘having many plans?’)”An AL’s report: “是对的, 应该.…… 我觉得解释应该是有很多计划. (wish you a happy voyage” vs. 一帆风顺 one-sail-wind-smooth “wish everything go smoothly”). Learners also associated a similar expression in their L1. The association strategy suggests that learners store their FS knowledge in a network fashion . When employing this strategy, both NSs and the two groups of learners would associate synonyms or antonyms, but only learners confused the target FS with another Chinese FS with a similar form, but not exactly the same meaning  was legitimate to be used in isolation. Metalinguistic analysis is often considered evidence of accessing explicit knowledge  and non-idioms (13.6%). NSs employed metalinguistic strategies less than 1% of the time, and SLs used zero metalinguistic strategies. By scrutinizing the think-aloud reports, we found that NSs’ metalinguistic cases often involved questioning whether a lexical bundle ”An AL’s report: “应该是对的, 因为谈和说有关的, 天和地有关的, 但是怎么用……不知道. ”This study examined the processing of Chinese idiomatic and non-idiomatic FSs among three groups of participants with different levels of proficiency. Due to the relatively small sample size, the findings should be considered to be tentative answers to our three research questions.This question aimed to compare AL, SL, and NS processing patterns through dichotomous judgment data. The analysis of the dichotomous judgments returned significantly different processing patterns among the three groups. NSs judged idioms more successfully than they judged non-idioms. ALs judged non-idioms more successfully than they judged idioms. SLs judged non-idioms slightly more accurately than they judged idioms, but the difference was marginal. The length effect was absent in all three groups. These patterns suggest that idioms are more deeply entrenched in NSs’ long-term memory than non-idioms and are thus unlikely to be recognized as wrong. For ALs, both types of FSs might not have been fully acquired, but since non-idioms are semantically and syntactically transparent forms, their correctness is easier to judge than idioms. SLs were almost equally successful with idioms and non-idioms, indicating that in the super-advanced stage, learners’ idiomatic knowledge improved compared to that in the advanced stage.This question aimed to compare AL, SL, and NS processing patterns through reaction time data. For NSs, idioms were processed significantly faster than non-idioms, regardless of the length of the FSs. In the AL group, we observed a reversed pattern; non-idioms were processed significantly faster than idioms were. Longer FSs took a significantly longer time to process than shorter ones did. For SLs, idioms were processed as fast as non-idioms, but longer FSs still required significantly longer processing times than shorter FSs. The presence of a length effect in the AL and SL groups suggests that learners were likely to read both idioms and non-idioms in a word-by-word fashion. For ALs whose idiom knowledge was quite limited, judging the correctness of an idiom may have largely relied on the online computation of the constituent words. Because idioms are semantically non-transparent forms, computing and making sense of an idiom would require more time than computing and making sense of a transparent non-idiom. However, SLs who possess better idiom knowledge still need to read an idiom word-by-word in the initial stages, but after they have reached the idiom key—the constituent word that determines when an idiom form can be recognized as an idiom —they areThis question aimed to compare AL, SL, and NS processing patterns through think-aloud data. We coded think-aloud statements for five levels of processing strategies. The frequency analysis showed that the two learner groups used different processing strategies from the NS group. NSs preferred to use the example strategy, while learners preferred to use the interpretation strategy. This pattern suggests that the nature of NSs and learners’ formulaic knowledge may be fundamentally different. Being able to provide examples indicates that NSs’ formulaic knowledge has become proceduralized. Providing interpretations implies that learners’ formulaic knowledge might still be descriptive . In addiIn sum, the following processing patterns emerged from the analyses of processing accuracy, speed, and strategy. First, the participants’ processing accuracy and global processing speed increased along with their proficiency. Second, CSL learners’ idiom processing ability was generally lower than that of non-idiom processing ability, but they demonstrated an improving trend as their proficiency level increased. Third, CSL learners’ use of processing strategies did not change much as proficiency rose and demonstrated a categorical difference from NSs. Fourth, all three groups exhibited poorer productive idiom knowledge than productive non-idiom knowledge.In the analyses of processing accuracy and processing speed, we found that SLs’ processing patterns were significantly different from those of ALs. From the advanced to the super-advanced levels, the learners changed from being more successful at non-idiom processing to being equally successful at non-idiom and idiom processing, marking a transition toward NS performance (being more successful at idiom processing). This finding suggests that formulaic knowledge can grow substantially as learners move from an advanced to a higher proficiency level.In the SLA literature, higher-than-advanced learners have long been overlooked. A major reason for this is that the number of achievers is very small, especially for learners of a non-English language such as Chinese. Another reason may have to do with the general assumption of the lexical plateau  with idioms than with non-idioms. Poor idiom competence was also reported by As for why using idioms is problematic, there are multiple reasons for learning difficulties. The primary one is the semantic opaqueness of idioms. Because of semantic and syntactic idiosyncrasies, learning idioms largely depends on rote memorization , which cAnother finding we would like to consider is the processing strategy used by the three groups of participants. Although the processing accuracy and speed data demonstrate a trend whereby L2 learners’ FS knowledge increased as they moved from the advanced to the super-advanced levels, the learners’ processing strategy pattern basically remained unchanged. While NSs used the example strategy the most, both ALs and SLs employed the interpretation strategy the most. The use of examples indicates that NSs can interact with FSs in a communicative manner, which represents a high degree of knowledge mastery. In contrast, the interpretation strategy involves describing or inferring FSs’ meaning; both cases suggest that CSL learners’ formulaic knowledge is descriptive and static . The queAlthough the experimental design enabled a controlled comparison of two types of formulae among three groups of Chinese speakers with different proficiency levels, a fact which had not been fully examined in the FS literature, we must acknowledge some limitations. First, the sample size of our study was relatively small; thus, the answers are more tentative than conclusive. Although higher-than-advanced learners are very scarce, endeavors could be made to conduct a larger-scale study.Another issue is the homogeneity of the FSs. In this study, we could subdivide both idioms and non-idioms. For example, non-idiomatic FSs included lexical collocations and lexical bundles. According to Regarding the methodology of think-aloud protocols, there may be the issue of reactivity; i.e., the possibility that thinking aloud may alter participants’ cognitive processes . AlthougAnother methodological issue concerns the use of NSs as the baseline group. According to our findings, NSs do not necessarily possess full idiom knowledge, especially productive idiom knowledge. Thus, future studies involving idiom production should carry out a preliminary test to confirm that the chosen NSs are eligible to serve as indexing norms.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by University of Illinois at Urbana-Champaign. The patients/participants provided their written informed consent to participate in this study.HZ designed the study, performed the experiments, analyzed the data, and wrote and edited the manuscript. BH analyzed the data, reviewed, and revised and edited the manuscript. JX supervised the research, analyzed the data, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The increasing burden of tuberculosis (TB) remains a very serious concern around the world, and account for a decreased quantity and quality of life. However, there is a limited epidemiology of the association of TB treatment with pregnancy. We aim to assess the effects of TB treatment in pregnancy complications.This will be a systematic review and meta-analysis of published studies on the association of TB treatment with pregnancy, retrieved from ScienceDirect, Web of Science, LILACS, Pubmed, Google scholar, Embase, Medline, ResearchGate, EBSCOhost and Cochrane library databases. The eligibility of the studies will be screened in accordance to the selection criteria by two independent reviewers. The quality and risk of bias of eligible studies will be performed by both reviewers using the Hoy tool and Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool in accordance to the measured outcomes . A data charting table will be used to extract background information and process the data items from each eligible study. The data will be analysed using Review Manager 5.3 (RevMan 5.3) software. Generic Inverse Variance method will be used for meta-analysis of both, individually and cluster randomized trials.The review and meta-analysis will not require ethical approval and the findings will be published in peer-reviewed journals and presented at local and international conferences. In addition, the study findings will be made accessible to the national committee of TB to formulate TB guidelines for their respective settings.International prospective Register of Systematic Reviews (PROSERO) number: CRD42021231872. According to Esmail et al the World Health Organisation estimated that about 2 million people have died from TB in the same year. The report is said to have characterized tuberculosis as the most infectious disease and the most leading cause of death around the globe. According to Van der Walt et al in 2018, South Africa was estimated to have had about 11 000 TB drug-resistant cases, of which 62% were estimated to be of Multidrug-resistant TB (MDR-TB). Van der Walt et al elaborated that the 62% estimate is driven by “Human Immunodeficiency Virus epidemic”.The burden of tuberculosis (TB) remains a very serious concern around the world. The World Health Organisation (WHO) mentioned that TB treatment during pregnancy differs due to immunological alterations induced by pregnancy. Therefore, latent TB infection can be treated using “Rifampin (RIF) (4R), Isoniazid (INH) and RIF (3HR), INH (6H/9H) with pyridoxine (Vitamin B) supplementation or INH and Rifapentine (3HP)” regime. However, INH and Rifapentine (3HP) is not recommended for pregnant women nor women expecting to conceive during TB treatment period due to lack of sufficient research on its safety during pregnancy. According to WHO guidelines TB disease can be treated using INH, Rifampin (RIF) and ethambutol (EMB), Streptomycin and Pyrazinamide (PZA). However, Streptomycin and PZA are not recommended because Streptomycin is mentioned to have shown harmful effects on the fetus whereas PZA's effects is not clearly defined. WHO further suggested HIV-related TB disease during pregnancy should be treated similar to a non-HIV related TB disease.TB can occur as latent TB infection, TB disease or HIV-related TB disease. Thus, treatment of drug-resistant TB (DR-TB) is still a challenge. Moro et al mentioned that the current recommended treatment is Isoniaze for nine months without delay. However, the updated WHO guidelines of (2016) suggests that drugs such as bedaquiline, delamanid, linezolid and fluoroquinolones are some of the most recommended drugs for DR-TB therapy.Van der Walt et al stated that pregnant women are often excluded during clinical trials, therefore, there exist no sufficient data to use of DR-TB treatment for pregnant woman. Therefore, HIV-co-infected pregnant woman are at risk of TB infection with TB prevalence of 1–11% in comparison to 0.06–0.53% of HIV-negative pregnant woman.TB pregnant women have a substantial burden, which have an estimated prevalence amongst pregnant and post-partum women starting from 0.06% to 7.2%. These estimates can reach as high as 11%. Bekker et al mentioned that HIV-infected pregnant woman were having a prevalence of 5.67 times more with a likelihood to have extrapulmonary TB. During pregnancy, T-helper (Th-1/ Th-2) cells have showed to be reduced, and these predominantly increases susceptibility rate to new infections. Therefore, this contributes to TB reactivation.,10 During early stages of post-partum period, Th-1 cells suppression may be reversed. This reversal might be due to the exacerbation of symptoms akin to the immune reconstitution syndrome. Research has however showed that tuberculosis during pregnancy can also be related to poor outcomes such as “perinatal death, stillbirths, premature birth, low birth weight (LBW), an increased risk of preterm birth, intrauterine growth restriction and hyaline membrane disease”.,10 TB is mentioned to be the most common cause of maternal deaths amounting to more than 35% of all maternal deaths. However, maternal death is mentioned to occur mostly in HIV-co-infected pregnant women. Pregnancy complications of exposure to TB treatment is not well known, however TB treatment in HIV co-infected and non-HIV co-infected pregnant woman yields a positive result. The study by Denti et al mentioned that pregnancy triggers physiological changes that has a major impact on the “absorption, distribution, renal or hepatic clearance and metabolism” of TB drugs during therapy. Findings from this study further mentioned that Rifampin is the major sterilizing drug in the therapy regime of drug sensitive TB and Azithromycin the precursor of pregnancy-induced cholestasis. Research showed that Isoniazid TB therapy during pregnancy had the ability to induce congenital abnormalities known as “talipes equinovarus” in an infant.TB positive pregnant woman tend to be more vulnerable, even during a post-partum period. However, due to teratogenic conditions, treatment can often be delayed to second trimester. Furthermore, Beck–Friis et al mentioned that there are only few studies conducted on tuberculosis treatment during pregnancy and pregnant women often experiences severe hepatotoxicity unlike non-pregnant woman. Therefor it is of paramount significant to closely monitor drug-resistant TB treatment in pregnancy to evade complications. In support to Esmail et al, Beck–Friis et al mentioned that liver transaminases must be monitored carefully during the treatment of TB in pregnant woman.,13 Furthermore, increase in enzymes of the liver must be observed and temporary withdrawal of drugs must be considered if there is an increase of enzymes. In these regard, the main objective of this review is to investigate the association of TB drugs among TB positive pregnant women.Esmail et al advised that drug-resistant TB treatment and its construction regimen during pregnancy should be in accordance to gestational age of the foetus and teratogenic anti-TB treatment effects should be weighed.2What might be the association between TB treatment and pregnancy complications?31.To assess the effects of TB treatment in pregnant woman.2.To assess the association of TB with pregnancy3.To examine TB treatment complications in pregnant women.4.To investigate the association of TB drugs among TB positive pregnant women.4 The results will be reported based on the PRISMA 2015, statement and article screening and selection process will also be demonstrated through a PRISMA-P flow diagram. Furthermore, the current protocol has been registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42021231872.This will be a systematic review and meta-analysis of published studies. This protocol is written in line with the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-analysis for Protocols (PRISMA-P) guidelines 2015.55.1This systematic review and meta-analysis will include randomized control trials, cohort, and matched cohort with a clearly defined population and interventions used. While, observational studies, reviews, case studies, and animal studies will be excluded in this study.5.2TB positive pregnant women.5.3Tuberculosis treatment.5.4TB negative pregnant women.5.5Hypertension in pregnancy, hypertensive disorders of pregnancy, pre-eclampsia, fetal growth restriction, miscarriage, recurrent spontaneous abortion.5.61.Evidence published in the English language.2.Evidence published between the years 2010–2020.3.Evidence from published global Randomized control trial, matched cohort, and cohort studies of TB treatment related pregnancy outcomes/ complication.4.All of the criteria defining the impact of TB treatment on the incidence of adverse pregnancy and foetal outcomes.5.71.Study which does not have the outcomes of interest as objectives.2.Case reports, expert opinions and review/meta-analysis.3.Evidence published before the year 2010.4.Evidence from TB non-pregnant women will be excluded because the impact of antidepressants is expected to be evaluated in pregnant women.5.8The following databases will be searched for eligible studies: Science direct, Medline, Embase, Pubmed, Africa Wide, Google scholar, ResearchGate, EBSCOhost, Web of Science, and the Cochrane Library, and LILACS. Medical subject headings (MeSH) such as “(((((((((((((TB[All Fields] AND positive[All Fields] AND (“pregnant women”[MeSH Terms] OR (“pregnant”[All Fields] AND “women”[All Fields]) OR “pregnant women”[All Fields])) AND ((“tuberculosis”[MeSH Terms] OR “tuberculosis”[All Fields]) AND positive[All Fields] AND (“pregnant women”[MeSH Terms] OR (“pregnant”[All Fields] AND “women”[All Fields]) OR “pregnant women”[All Fields]))) AND (TB[All Fields] AND (“therapy”[Subheading] OR “therapy”[All Fields] OR “treatment”[All Fields] OR “therapeutics”[MeSH Terms] OR “therapeutics”[All Fields]))) AND (TB[All Fields] AND negative[All Fields] AND (“pregnant women”[MeSH Terms] OR (“pregnant”[All Fields] AND “women”[All Fields]) OR “pregnant women”[All Fields]))) AND  OR “pregnancy-induced hypertension”[All Fields] OR (“hypertension”[All Fields] AND “pregnancy”[All Fields]) OR “hypertension in pregnancy”[All Fields])) OR  OR “spontaneous abortion”[All Fields] OR “miscarriage”[All Fields])) OR (hypertensive[All Fields] AND (“disease”[MeSH Terms] OR “disease”[All Fields] OR “disorders”[All Fields]) AND (“pregnancy”[MeSH Terms] OR “pregnancy”[All Fields]))) OR (“pre-eclampsia”[MeSH Terms] OR “pre-eclampsia”[All Fields] OR (“pre”[All Fields] AND “eclampsia”[All Fields]) OR “pre eclampsia”[All Fields])) OR  OR “fetal growth retardation”[All Fields] OR  OR “fetal growth restriction”[All Fields])) OR  OR “spontaneous abortion”[All Fields] OR “miscarriage”[All Fields])) OR  OR “spontaneous abortion”[All Fields] OR (“spontaneous”[All Fields] AND “abortion”[All Fields])))) AND  OR “random allocation”[All Fields] OR “randomized”[All Fields]) AND (“prevention and control”[Subheading] OR (“prevention”[All Fields] AND “control”[All Fields]) OR “prevention and control”[All Fields] OR “control”[All Fields] OR “control groups”[MeSH Terms] OR (“control”[All Fields] AND “groups”[All Fields]) OR “control groups”[All Fields]) AND )) OR (“cohort studies”[MeSH Terms] OR (“cohort”[All Fields] AND “studies”[All Fields]) OR “cohort studies”[All Fields] OR “cohort”[All Fields])) OR (matched[All Fields] AND (“cohort studies”[MeSH Terms] OR (“cohort”[All Fields] AND “studies”[All Fields]) OR “cohort studies”[All Fields] OR “cohort”[All Fields])) and free text searches will be used to search the eligible articles which will be saved to the citation manager Zotero v5.0.81 (Zotero.org). This software will also be used to remove duplicates. The title and abstracts of the articles remaining after exclusion of duplicates will be assessed for eligibility according to the inclusion and exclusion criteria.5.9The full text of all potentially eligible studies will then be reviewed by two independent reviewers (MAK and WNP), and any disagreement between reviewers with respect to eligible studies for inclusion in the analysis will be assessed for more eligible studies. Initially, studies will be screened by the titles, abstracts, keywords, and synonyms then followed by the identification of the full-text articles. Should discrepancies arise between 2 authors , a third author will screen such studies, and consensus will be reached through discussion. Zotero v5.0.81 (Zotero.org) will be used to manage extracted data items, including saving relevant and excluded studies with reasons. Importantly, reference lists of included studies will be screened to confirm that no relevant studies are left out. Studies meeting the inclusion criteria will then be subjected to data collection, critical appraisal, risk, and quality evaluation.66.1The reviewers (MAK and WNP) will develop a data extraction form that will be used in the collection relevant data items. To reduce data entry errors, selected studies will be independently assessed by two reviewers (MAK and WNP), the third reviewer will be consulted for arbitration in case of any disagreements.6.2Extracted data items will include the author's name, year of publication, HIV status: (report percentages + and -), TB status: (report percentages + and -), HIV treatment, TB treatment, gestational age, adverse maternal outcomes: Hypertensive disorders of pregnancy, pregnancy induced hypertension, pre-clampsia, adverse fetal outcomes: Fetal growth restriction, miscarriage, recurrent spontaneous abortion.6.3 and Downs and Black checklist will be used. Two independent reviewers (MAK and WNP) will appraise all included studies and a third reviewer will be consulted in cases of disagreements.To evaluate the potential risk of bias in randomized control trials, cohort, and matched cohort, Cochrane collaboration tool for assessing bias6.42 and Chi squared statistical tests will be used.,18 An I2 value of > 50% will be considered substantial heterogeneity. To find the sources of heterogeneity within the included studies, a subgroup analysis and meta-regression comparing the study estimates from different study-level characteristics, quality, intervention type, and the reported effect measure of adverse events will be conducted.A summary of findings table will be used to provide a synthesis of the main outcomes of included studies. data will be analyzed with Rev Manager (Version 5.3) to conduct a meta-analysis. To measure statistical heterogeneity between studies, I7 will be used to assess the overall quality of evidence. Moreover, the quality of each included study will be independently evaluated by 2 authors . The third author will adjudicate in cases of disagreements. The quality of evidence will be assessed based on several factors such as study limitations, indirectness of results, and publication or reporting bias. The evidence of each outcome will be rated as high, moderate, low, or very low.The Grading of Recommendations, Assessment, Development and Evaluation assessment tool8 Therefore, this systemic review and meta-analysis aimed to investigate the effect of TB treatment on pregnancy complications. Findings from this study will help to give a better understanding on the pathophysiology of TB in pregnancy complications as wells as to understand the effect of TB treatment in pregnancy complications in order to improve future diagnostic criteria's, thereby reducing maternal and fetal adverse outcomes.Tuberculosis (TB) infection during pregnancy has been reported to be associated with maternal and fetal adverse effects and has not been critically elucidated.Conceptualization: Wendy Phoswa, Moloko Adolph Kgathi.Investigation: Wendy Phoswa, Moloko Adolph Kgathi.Methodology: Wendy Phoswa.Resources: Wendy Phoswa.Software: Wendy Phoswa.Supervision: Wendy Phoswa.Validation: Wendy Phoswa, Moloko Adolph Kgathi.Visualization: Wendy Phoswa.Writing – original draft: Wendy Phoswa, Moloko Adolph Kgathi.Writing – review & editing: Wendy Phoswa, Moloko Adolph Kgathi.


The macroscopic mechanicalproperties show ductile-to-brittle change with an increase in theclay volume fraction from 54% to 70%. Conceptually, this work revealsthe ultimate elastic and thermal properties of aligned layered claynanocomposites in flaw-tolerant conditions.Layerednanomaterials fascinate researchers for their mechanical,barrier, optical, and transport properties. Nacre is a biologicalexample thereof, combining excellent mechanical properties by alignedsubmicron inorganic platelets and nanoscale proteinic interlayers.Mimicking nacre with advanced nanosheets requires ultraconfined organiclayers aimed at nacre-like high reinforcement fractions. We describeinorganic/polymer hybrid Bragg stacks with one or two fluorohectoriteclay layers alternating with one or two poly(ethylene glycol) layers.As indicated by X-ray diffraction, perfect one-dimensional crystallinityallows for homogeneous single-phase materials with up to a 84% clayvolume fraction. Brillouin light spectroscopy allows the explorationof ultimate mechanical moduli without disturbance by flaws, suggestingan unprecedentedly high Young’s modulus of 162 GPa alongthe aligned clays, indicating almost ideal reinforcement under theseconditions. Importantly, low heat conductivity is observed acrossfilms, κ A classic strong and toughbiological composite is pearl of nacre, which consists of a high volumefraction (95 vol %) of aligned reinforcing inorganic aragonite platelets and a soft biopolymer layer(thickness 10–50 nm), which are hierarchically arranged ina so-called “brick-and-mortar” structure.5 In spite of extensive research,the reasons for the extraordinary mechanical properties are not fullyunderstood because of the complex combination of several mechanisms,hampering rational designs of advanced bioinspired materials. Grosslyoversimplified, it has been postulated that the ceramic componentprovides reinforcement, whereas the biopolymer enables the distributionof localized stresses and provides fracture energy dissipation. However,the actual behavior is much more complex and also involved mineralbridges between the reinforcements.5Biological compositeshave attracted growing interest because theyare concomitantly strong and tough and have low density.in situ; otherwise, excessive packing defects remain. Ahigh volume fraction cannot be achieved using prefabricated, thick,solid, inorganic sheets. (2) By contrast, if nanometric sheets thatare strong in tension but flexible in bending are used, their packingdefects can be largely relieved, but a high reinforcement volume fractioncannot be easily achieved unless the polymer layers are ultrathin.Both approaches have been adopted toward nacre-inspired materials,ranging from in situ mineralization to the use ofvarious nanosheets.20 The latter approach has been widely exploited because it may allowstronger reinforcements than those when relatively brittle ceramicsare used. Nanosheets can also serve other functions, such as improvingthe gas barrier properties24 or incorporating electrical conductivity by using,for example, mXene.25What is significant in nacre is the considerable thickness of ca.10–50 nm of the macromolecular layers, which, in turn, requiresa large thickness of the reinforcing platelets, while assuring a highvolume fraction of reinforcements. It has been conceived that, inthis way, the dynamic polymer chains can promote the fracture energydissipative transformations. Two opposite approaches can be consideredfor nacre-mimetic designs: (1) If thick submicron inorganic layersare really needed to concomitantly allow for a high filler contentwhile keeping the thick polymer layers, they have to be biomineralized 20 andsome approaches have shown even nacre-like toughness in notched fracturemechanical studies,27 composite elastic moduli have typically remained much smaller thanexpected from the constituents. For example, the nacre-mimetic materialsbased on clay typically show Young’s modulus (E) values of some tens of gigapascal (GPa), while pure clay nanosheetsshow E values of 146–171 GPa.29 Even complex combinations of graphene modifications, clay, and carbonnanotubes show only a modest E modulus value of 130GPa,20 signifying a fundamental problemof transferring the reinforcement elastic properties to the compositesystem. The question remains as to whether the low modulus value isdue to insufficient stress transfer across the interfaces or due todefects. A second question relates to using nanometric two-dimensional(2D) nanosheets, of which several types have been used, such as clays,graphene and its modifications, mXenes, and titanates: Could a highvolume fraction of reinforcements still be achieved in cases thatuse an organic “matrix”? Also, what are the mechanicalproperties, in particular the elastic moduli, of the composite systems,considering the ultraconfinement of the polymer layers? In addition,if such properties are achieved, does the presence of elastic anisotropyindicate the presence of thermal anisotropy? Finally, how much doesdefect tolerance affect the macroscopic mechanical properties?Although approacheshave been provided to allow a high ultimatestrength in nacre-mimetic materials0.5]inter[Mg2.5Li0.5]oct[Si4]tetO10F2), which delaminates based onthe rare phenomenon of repulsive osmotic swelling, yielding an unprecedentedthickness homogeneity and complete separation.31 This contrasts the standard liquid-phase exfoliation of classicallyused natural clays, such as montmorillonite, which only provides abroad distribution of thicknesses and poorly defined materials. Importantly,osmotic swelling produces a nematic liquid-crystalline phase thatcan be homogeneously mixed with a polymer.Herein we address these questions by constructing one-dimensional(1D) crystalline arrangements (“hybrid Bragg stacks”)of nanosheets and polymer layers with a superior translational periodicity.We use synthetic sodium fluorohectorite (inter[Mg2.5Li0.5]oct[Si4]tetO10F2, obtained by melt synthesis,41 followed by long-term annealing,38 shows such a rare phenomenon. Additionally, it possesses phase purity,homogeneous charge density, and thus uniform intracrystalline reactivity.Because of its exceptional charge homogeneity, the as-synthesizedfluorohectorite can be transformed into an ordered interstratificationby partial ion exchange with NH4+. Na+ and NH4+ ions segregate to different interlayers,and the two types of interlayers strictly alternate in this orderedheterostructure, which represents a thermodynamically stable state.Because of the lower hydration enthalpy of NH4+, hydration in this interlayer type is completely blocked.Among the2D materials, only a handful of compounds are known toshow repulsive osmotic swelling38 This suspension is not isotropic but represents a nematicphase, where the nanosheets are held in a cofacial arrangement becauseof their electrostatic repulsion. When the heterostructure (selectiveNa+ exchange to NH4+ in every otherinterlayer) is immersed in water, every other interlayer type is blockedfrom osmotic swelling, and it, consequently, spontaneously delaminatesinto 2-nm-thick nanosheets (DS),29 wheretwo hectorite layers are connected via a nonswelling interlayer ofNH4+ ions. To obtain the most frequently usedmontmorillonite platelet diameter , ultrasonicationis applied to reduce the equivalent diameters of Hec and DS to 340and 406 nm, respectively . Innacre, the macromolecules in the organic layers are particularly soft;5 therefore, PEG with a low glass transition temperature(Tg = −20 °C) was selected.Being water-soluble and nonionic, it does not interfere with electrostaticstabilization of the nematic suspensions. Moreover, PEG is known toform complexes with Na+,42 and,consequently, intercalated phases with two different PEG volumes maybe obtained36 because phase separationis counterpoised by interaction with the interlayer cation.When the pristine fluorohectorite is immersed in deionized water,individual 1-nm-thick nanosheets are obtained.31 Therefore, solution compounding could be achievedsimplyby mixing the nematic suspensions with different amounts of an aqueousPEG solution, which preserves the nematic character of the suspension,as is evidenced by small-angle X-ray scattering . Spray coating onto poly foils, followed by gentle drying, produced transparenthybrid films  suspensions represent nematic phases in water, withthe nanosheets separated by large distances (>50 nm).ng SAXS; S2. Sprayng SAXS; S2. Spray44 and 30 vol %36 corresponding to periodicities of 1.8 and 1.4 nm, respectively.The present observed periodicities . Our samples with optimized PEGcontents feature intense basal reflections of a rational 00l series of up to the fifth and fourth order and a verylow coefficient of variation (CV) of the 00l series. As expected, the fwhm minima  at 54 vol % (Hec/2PEG) and 70 vol% (Hec/1PEG) of Hec coincide with the CV minima . Such low CVs imply extraordinarily uniform interlayerheights, which usually require ionic or molecular interlayer cationsof the same type and well-defined size and shape. Observing such alow CV for a hybrid Bragg stack with intercalated macromolecules isquite surprising because even low-molecular-weight polymers are intrinsicallypolydisperse and thus are expected to adopt different conformations.Previous studies have reported intercalated phases of layeredsilicateswith PEG volume contents of 46 vol %d spacings increase by 1 nm upon going from Hec to DS forDS/2PEG and DS/1PEG , correspondingto the thickness of a hectorite nanosheet because NH4+ fits into the surface corrugations and does not require additionalspace. The quality of the 1D crystallinity of the DS Bragg stacksis comparable to that of the Hec Bragg stacks, as indicated by theintense reflections, and a rational 00l series visibleup to the ninth and seventh order for DS/2PEG and DS/1PEG, respectively, while the volume contentis the same within experimental errors (Table S1). As shown by the X-ray diffraction (XRD) patterns and TEMimages, the periodicity of DS/2PEG is doubled, and thus the interfacedensity between PEG and the clay is halved. We use these four distinct,transparent samples with ultimate structural control, a maximum fillercontent, and polymer confinement to test the intrinsic, direction-dependentthermal and mechanical properties. We include in our analysis an alternativepolymer, PVP, which is also capable of forming distinct, crystallinehybrid Bragg stacks with 31 and 40 vol % of Hec.32Similar to the Hec-based films, the periodicity and filler contentof the films could be varied systematically at discrete steps by applyingthe DS filler without compromising the homogeneity or quality of the1D crystallinity. As described for Hec/2PEG and Hec/1PEG, the PEGcontent was carefully optimized, and the optimum 1D crystallinitywas observed for 70 and 82 vol % of DS fillers with CV values of 1.6and 1.0 % for DS/2PEG and DS/1PEG, respectively 2, bottomectively2, bottomectively2, middleq. The magnitude, q, depends on the incidentlaser wavelength, λ = 532 nm, the scattering angle, θ,and the material refractive index, n (section S3). Hence, the probed phonon wavelengthvaries in the range of about 200–2000 nm.BLS hasimportant advantagescompared to macroscopic mechanical experiments: (i) measurement ofthe elastic moduli under zero strain, utilizing thermal phonons inmaterials, (ii) determination of the elastic tensor components, probingdirection-dependent phonon propagation in anisotropic materials, and(iii) insensitivity to material defects due to the submicron resolutionof the BLS technique, ensuring determination of the ultimate materialelasticity, which should be the ultimate goal of defect engineering.BLS results from the inelastic scattering of light by the longitudinaland transverse phonons propagating in the medium with a wave vector f = ±cq/2π around the central Rayleighline, with c being the longitudinal (cL) or transverse (cT) soundvelocity that determines the corresponding longitudinal, E = 2G(1+ v) moduli, with ρbeing the density and Tg < T < Tg + 100 K, where Tg is the glass transition temperature, the moduli aretruly elastic .46For elasticallyisotropic media, the BLS spectrum is Doppler shifted at 32 and soft materials (section S4).48 However, for an unambiguousdetermination of the elastic constants, full utilization of the BLSgeometries and polarizations is needed. We performed measurementsat three different scattering geometries—transmission, reflection,and backscattering—to probe phonon propagation along the directionsparallel, normal, and oblique to the present Hec/PEG films, respectively(insets to section S3). Polarizationof the incident and scattered light was selected to be either vertical(V) or horizontal (H). Different polarization configurations  allow for selective examination of the existing modes (section S4).For elastically anisotropic materials like the hybrid Braggstacksin this work, determination of the stiffness constants of the elastictensor requires access to the direction-dependent polarized and depolarizedBLS spectra. The methodology has recently been reported for severalhard∥ andT∥ modes. However, we also observe additional modesfrom backscattering contributions due to the internal reflection ofthe laser beam on the sample’s backside (right inset to 50⊥) and a single out-of-plane transverse mode (T⊥) are present in the VV and VH spectra, respectively. Note that thelatter VH spectrum is much weaker than the corresponding in-planemode . We started from the transmission geometry, for which the magnitudeof q∥ is independent of the refractiveindex n. The dispersion relation f(q∥) for both L∥ and T∥ is linear  and, hence, c(α = 90°) for both phononsis q-independent (see section S3 for details). To determine q⊥ at the reflection geometry and qbs atthe backscattering geometry, the refractive index of the sample hasto be known. We have thus recorded the dispersion f(q⊥), which was forced to be linearthrough the origin , to determinethe value of n. In this way, we obtained the soundvelocities c(α = 0°) for both out-of-planemodes (L⊥ and T⊥). For the oblique q directions in the backscattering measurements .32 Because of the relationship betweenthe elasticity tensor components and sound velocities (eqs S1–S7), a theoretical framework ofthe direction-dependent sound velocities was obtained. The sound velocitiesof the Q–L, Q–T, and P–T modes are shown in Figure S8. The reflection and transmission measurements yield cQ–L and cQ–T at α = 0° and cQ–L and cP–T at α = 90°,respectively. Note that the backscattering measurements address allthree modes  at intermediateα angles, which are limited below about 50° by the sample’srefractive index.For a transversely isotropicmaterial, the elastic stiffness tensorcontains five independent elastic constants, which we determined throughχE∥), cross-plane Young’smodulus (E⊥), sliding shear modulus(G13), torsional shear modulus (G12), bulk modulus (K), andtwo characteristic Poisson’s ratios  and the out-of-planeYoung’s modulus as E⊥ (andnot E33). The exact values of the elasticstiffness constants and engineering moduli are summarized in Tables S3 and The theoretical representation of the soundvelocities also allowedfor designation of the engineering moduli 3e,f. ThemodulusG3, torsio52 Forthe in-plane moduli, it is a linear interpolation of the volume-weightedelastic properties of the individual layers, as is also implied bythe experimental E∥ in E∥(ϕHec) is well represented bya linear regression using E∥,Hec = 179 GPa and EPEG = 6.1 GPa, suggestinga weak impact of the predominantly parallel Hec/PEG interfaces; EPEG = 6.1 GPa is a meaningful value for glassyor crystalline polymers in a stretched conformation. The large elasticitymismatch between the Hec and soft polymer layers obscures any conclusionon the polymer specificity (PVP vs PEG) on E∥. Furthermore, the conformity of E∥ to the predictive mixing rule suggests that E∥ is an inherent material property thatshould monotonically depend on the filling ratio.Depending on thedirection, a systematic variation of the elasticmoduli with the composition is displayed in E⊥.53 However, as previously reported for Hec/PVPBragg stacks,32E⊥ because the predicted dependence isexhibits a concave form  incontrast to the experimental convex form. For the high filling fractions(>50%), we find a plateau region with E⊥ comparable to that at 100% Hec. The very high filler contents appliedhere cause a severe confinement of the thin polymer layers. As a consequence,the polymer interlayers fail to efficiently decouple adjacent nanosheetsin the stack, which would be required for energy dissipation.Along the1D periodicity direction, the phonon dispersion relationshipat long wavelength predicts Wood’s law for the compositiondependence of the cross-plane modulus E⊥ is unaffected by the presence of a confined polymer,whereas at <50 vol % Hec, the increasing volume fraction of PVPdoes lead to a decreasing E⊥ butstronger than the Wood’s law prediction . The clearly different composition dependence of E⊥ and E∥ emphasizes the role of the interface density solely for the cross-planeelasticity (above ∼50 vol % Hec). Because of this difference,the elasticity anisotropy, E∥/E⊥, increases with the Hec content from4.6 at 54 vol % Hec to 6.7 at 82 vol % Hec  increase by about 50% as theHec content increases from 54 to 82 vol %. The bulk modulus K increases only by about 15% and is comparable to E⊥ with a similar saturation above ∼60vol % Hec because both are primarily controlled by the elastic constant C33. Consistently, the cross-plane moduli, E⊥ and G13, are hardly influenced by the composition  for Hec/PEG than Hec/PVP hybrid Bragg stacks. This is the onlyindication of a polymer specificity that we see in the two investigatedsystems. We attribute this to the more compact PEG intercalation betweenthe Hec sheets with less free volume.Analogousto behavior 3e,f.Conposition 3e,f. The12 represents how the sample shrinksin the “2” direction due to an applied stretching strainin the “1” direction. Note that the “1–2”plane means the sample plane, and hence the high ν12 value is polymer-like. The second Poisson’s ratio ν31 expresses how the sample expands in the “1”direction due to an applied compressing strain in the “3”direction. Hence, the almost zero value of ν31 impliesnegligible deformation of the PEG layer along the “1”direction; we note that wine corks have a Poisson’s ratio ofnearly zero. Among the different elasticity parameters, G13 and ν12 reflect the properties ofthe confined polymer.The Poisson’s ratio1 ν12 rep56 Analogous to the BLS characterization, we include the previouslyreported Hec/PVP system with one (40 vol % Hec) and two (31 vol %)layers of PVP, respectively. The in-plane thermal conductivity κ∥ follows a linear composition dependency. Accordingto the parallel thermal resistance network model, κ∥ = κPEG + (κHec(DS) – κPEG)ϕHec, with κHec(DS) andκPEG being the thermal conductivities of Hec (orDS) and PEG, respectively, and ϕHec the Hec volumefraction  in the Hec/PVP system.32 According to the serial thermal resistance networkmodel, 1/κ⊥ = ϕHec +(1/κPEG), with RHec/PEG and d⊥,Hec being the thermalresistance per unit area of a Hec/PEG interface and the thicknessof one Hec layer, respectively. The increasing κ⊥ with ϕHec then suggests that −1/κPEG + 1/κHec + 2RHec/PEG/d⊥,Hec < 0. Therefore, thecross-plane thermal resistances of the Hec/PEG samples are not dominatedby the Hec/PEG interfaces, unlike the situation of the Hec/PVP samples,where the stronger influence of the interfaces leads to a plateauof κ⊥ in the considered Hec volume fractionrange. The different effects of the Hec/PEG and Hec/PVP interfacescould stem from the different physical states of PEG and PVP becauseof the vastly different glass transition temperatures (210 vs 445K) and the stronger complexation between PEG and Na+.42 Both effects can influence the chain mobilitywithin the confined space and, consequently, lead to a different interfacialadhesion between the Hec/PEG and Hec/PVP interfaces, respectively.In the case of PEG, the decrease can also be driven by the introductionof different types of interfaces in the hybrid material. While thesamples with 1PEG involve only polymer–hectorite interfaces,for 2PEG an additional polymer–polymer interface is introduced(if a stretched PEG conformation is assumed). One should further notethat κ⊥ is more strongly reduced for DS thanfor Hec nanosheets. We attribute this to an additional type of interfacecharacteristic for the DS filler, namely, the NH4+ cation interlayer “gluing” two Hec sheets to forma DS.Because of the large thermal conductivity contrast between thepolymers and Hec/DS, we do not see a polymer-specific behavior. Thecross-plane thermal conductivity κ31 vol% 4b, which∥, E∥, and G12, which is emphasized by the black dashedline ,a monotonic decrease with decreasing filler content can be observedin the case of κ⊥. This decrease is even moredramatic considering that κ⊥ of the hybridfilms is consistently lower than that of the pure components. We can,therefore, conclude that the interface chemistry has a significantcontribution to the cross-plane heat transport. As outlined above,the cross-plane thermal transport continuously decreases with theintroduction of additional types of interfaces . The cross-plane mechanical moduli (E⊥ and G13) are notinfluenced by these different types of interfaces. This also becomesapparent in the correlation plots (E⊥ versus κ⊥ exhibits a flat line (black dashedline) for filler contents of >50 vol %. This difference in sensitivityto the presence of the nanostructure and interfaces is further translatedinto a 4–5-fold increased anisotropy compared to the mechanicalanisotropy. The similar composition dependencies of κ⊥ and κ∥ result in a roughly constant anisotropyratio  are interestingly high because comparablevalues in nanocomposites are only achieved with high Tg polymers57 in combination with cross-linking58 or by combining low Tg polymers withhydrogen-bonding networks.59 Second, the correlationbetween E and ε (%) at different Hec or DScontents is not straightforward. In contrast to the other Hec/PEGhybrid materials, Hec/2PEG displays a plastic deformation at σ= 48 MPa and breaks at a high elongation of 8.4%  without breaking. Hec/2PEG features the smallest E value but is still comparable to the Hec/1PEG system,which has the smallest elongation at break (ε = 0.4%). Hec/2PEG,consequently, represents a unique and singular property that is elusiveto a predictive material engineering based on the effective materialproperties. This finding suggests that macroscopic stress–strainmeasurements alone are inappropriate to guide the structure propertyoptimization of nacre mimetics. Third, E does notfollow the expected increasing trend with the filler content. WhileDS hybrids are indeed stronger than the corresponding single Hec counterparts,DS/1PEG with 82 vol % Hec is weaker than DS/2PEG . The missing correlation of E with the filler content suggests that even in the linear-elasticregion at very low strain, nonlocal contributions, for instance, bydefects, are significant.The macroscopic mechanical propertiesare obtained from the stress–strain curves of the hybrid Braggstack films measured at 53% relative humidity, as shown in inset of 5a. This E∥, from BLS  was obtained via meltsynthesis followed by long-term annealing,according to an established procedure.41 The synthesisof ordered heterostructures (interstratifications) consisting of strictlyalternating sodium and ammonium interlayers was performed accordingto an already published procedure.29 PEG(Mw = 1500 g mol–1)was provided by Sigma-Aldrich.The synthetic clay [Na21 Consequently,we conducted all of our experiments at ambient humidity conditions,without further environmental control. Additional information aboutthe sample preparation and characterization methods can be found in sections S1 and S2.The as-synthesizedfluorohectorites and heterostructures were delaminated by immersingthem in Millipore water (0.4 vol %), producing nematic liquid-crystallinesuspensions of Hec (1 nm) and DS (2 nm), respectively. For a reductionof the original diameters of Hec and DS, the suspensions were sonicatedwith an ultrasonic horn. PEG was dissolved in Millipore water (0.8vol %) and added in the desired weight ratio. The suspension was mixedfor at least 1 day in an overhead shaker. The quality of the suspension,in terms of homogeneity, was crosschecked by SAXS. Self-supportingfilms were prepared by employing a fully automatic spray-coating system.Every spraying cycle was followed by a drying cycle of 90 s at a temperatureof 40 °C. The films were characterized by XRD, TEM, and thermogravimetricanalysis. The mechanical characteristics were determined by tensiletesting with a ZwickRoell testing machine equipped with a 20 N loadcell. In contrast to the pure components PEG and Hec, nanocompositeswith such high filler contents have been shown to be barely sensitiveto ambient relative humidities.q), by detecting the Dopplerfrequency shift, ω, ofthe inelastically scattered light by sound waves (“phonons”)with a wave vector, q. We recorded BLS spectra by utilizingthree scattering geometries and two polarization configurations of the incident (λ = 532nm) and scattered  light, which allowedus to establish the nature of the observed phonons. We varied theincidence angle to obtain the direction-dependent sound velocitiesnecessary for determination of the anisotropic elasticity. The elasticstiffness tensor was obtained from the representation of the direction-dependentsound velocities by the Christoffel equation assuming transverse isotropy.The characteristic Young’s moduli, shear moduli, and Poisson’sratios of the Bragg stacks were subsequently calculated. More detailscan be found in sections S3 and S4.BLS recordsthe phonon dispersion, ω, assuming 1D heat transfer. With thefilm thickness determined by laser scanning microscopy, the thermalconductivity was obtained, neglecting thermal contact resistances.More details are provided in