The development of information technology has brought great convenience to our lives, but at the same time, the unfairness and privacy issues brought about by traditional centralized systems cannot be ignored. Blockchain is a peer-to-peer and decentralized ledger technology that has the characteristics of transparency, consistency, traceability and fairness, but it reveals private information in some scenarios. Secure multi-party computation (MPC) guarantees enhanced privacy and correctness, so many researchers have been trying to combine secure MPC with blockchain to deal with privacy and trust issues. In this paper, we used homomorphic encryption, secret sharing and zero-knowledge proofs to construct a publicly verifiable secure MPC protocol consisting of two parts—an on-chain computation phase and an off-chain preprocessing phase—and we integrated the protocol as part of the chaincode in Hyperledger Fabric to protect the privacy of transaction data. Experiments showed that our solution performed well on a permissioned blockchain. Most of the time taken to complete the protocol was spent on communication, so the performance has a great deal of room to grow. Traditional centralized systems provide efficient and personalized service, but the negative effects of centralization are increasingly appearing: corruption, inequality and privacy issues. As it turns out, some decentralized technologies  are urgeA core problem is that all users who have joined the blockchain see an identical ledger, making it thorny to handle transactions that rely on confidential data . Access Excitingly, the cryptographic technology of secure multi-party computation is a perfect way to deal with the problem of privacy. This concept dates back to what is called “Yao’s millionaires’ problem” , a famoutransfer , garbledtransfer , homomortransfer  and the transfer ,10.Secure MPC provides enhanced privacy, correctness and independence of inputs, and guarantees output delivery. Blockchain perfectly suits secure MPC protocols because they all deal with security and trust issues in distributed environments . There aIn this paper, we propose a publicly verifiable, secure MPC protocol to protect the transaction privacy of a permissioned blockchain. The protocol contains two parts: an on-chain computation phase and an off-chain preprocessing phase. Operations such as key generation, data encryption and generating pre-processing data are implemented off-chain, and secure computations are performed on-chain. The execution of the on-chain protocol is integrated as part of the chaincode in Hyperledger Fabric . Concret(1) In the on-chain phase, we facilitate additive secret sharing and the Paillier cryptosystem, an additive homomorphism encryption scheme, to preserve the confidentiality of private data. Computations are based on encrypted shares and can be parallelized, which greatly resolves the N-1 attack problem. We integrated the on-chain protocol as part of the smart contract to utilize its correctness and verifiability. (2) We adopted zero-knowledge proof technology, specifically that of Pedersen , to consrom SPDZ ). (4) FiThe remainder of the paper is structured as follows. In Many works have been done to protect privacy in the blockchain in recent years. Frequently used cryptographic techniques  for privHere we mainly focus on investigating the development of the combination of blockchain and secure MPC. Bitcoin was first utilized to obtain fairness in a secure multi-party protocol ,25,26. OSánchez  considerSome researchers focused on Hyperledger Fabric, a typical permissioned blockchain hosted by the Linux Foundation. Benhamouda et al.  used secThe previous work can be classified into two main types: on-chain secure MPC protocols that integrate the protocols into the blockchain architecture itself, and off-chain secure MPC protocols that offload intensive computation to an off-chain network. Benhamouda  describeOur MPC-over-Fabric architecture is similar to the on-chain secure MPC protocol described by Benhamouda , but notIn this section, we describe the main framework of the proposed secure MPC computation scheme. Participant: the data owner.Peer: compute node in Hyperledger Fabric, concretely the endorsement node.Decryptor: a component in the Peer assisting decrypting during the endorsement phase. : All participants use the quadruple generation protocol to obtain a sufficient number of (On-chain computation phase): All participants break raw input values to secret shares, encrypt secret shares and quadruple shares, generate commitments and submit all of them to the ledger.(On-chain computation phase): Participants store the addresses of decryptors to the ledger.(On-chain computation phase): The invoker (one of the participants) invokes the MPC smart contract to execute the computation task.(On-chain computation phase): Participants decrypt their outputs, which can also be carried out by decryptors, and reconstruct the final result.A jointly secure computation task proceeds according to the following steps:In this section, we describe the cryptography algorithms used in our protocol.(1)Additive homomorphism encryptionAdditive homomorphic encryption allows one to perform computations on ciphertext. The result of the calculation is the encryption of their sum.KeyGen: takes the message Dec: takes the ciphertext Add: takes two ciphertexts Sub: takes two ciphertexts cMul: takes the scalar Let AHE =  be an instance of an asymmetric additive homomorphism encryption scheme. We will use the following definitions: The message space is denoted as N is the security parameter.In this paper, we use the popular Paillier  cryptosy(2) Additive secret sharingn,n)-threshold additive secret sharing scheme and its implementation is as follows [In this paper, we use ( follows .p), and computes Secret shares: To share a secret a, the dealer chooses Secret reconstruction: All participants collaboratively reconstruct the secret: a and b, the shares of The above implementation possesses additive homomorphism. Concretely, if participants (3)PedersenE over a field Setup Phase: All participants agree on an elliptic curve r is a blinding factor that prevents observers from guessing Commitment Phase: Participant Open Phase: Pedersen ,34 is a The Pedersen algorithm also has additive homomorphism and it is easy to prove the following equations:Privacy confidentiality: All the input values and output values are in encrypted form and no one learns anything except his own input secret and output values.Publicly verifiable: Computations can be publicly executed and are controlled by no one. Any step during computation can be publicly verified.In this section, we present the core idea of our secure MPC protocol. Since a blockchain is completely public, we aim to build a protocol for multi-party arithmetic computation over Similarly to SPDZ , structuPTP), which can be instantiated with a trusted execution environment (TEE) or a trusted player. In this paper, the smart contract in the blockchain plays the role of PTP. PTP receives inputs, calculates functions and generates outputs without mistakes. All behaviors of PTP are public and can be verified.We assume a publicly trusted processor Protocol InputAll participants Each participant n random blinding factor PTP.Finally, PTP and decrypt Other participants If true, the share PTP before computation. PTP:Moreover, participants must submit enough quadruples (2)Protocol AdditionAn addition gate is defined as follows:n participants. The i-th output value The inputs are the encrypted shares of secret inputs PTP computes:To compute PTP computes:For a public scalar PTP computes:To compute n,n)-threshold additive secret sharing and the Paillier cryptosystem are additively homomorphic, any linear function with n inputs can be computed locally without interactions.Since both .Note that we do not need open commitment after every addition gate, resulting from the fact that the addition gate can be automatically operated by (3)Protocol MultiplicationA multiplication gate is defined as follows:n participants. The i-th output value The input is the encrypted shares of secret inputs a, b and c are random finite field elements unknown to everyone, and d equals 1. d is necessary because it makes it possible to perform Beaver Multiplication [Multiplication consumes  triples . The baslication  in encryObtain the encrypted shares of t, s and Obtain Obtain the encrypted shares of To compute Now we have:Therefore, the result is correct.m, then the depth of the multiplication gate is To reduce the communication complexity and improve the computation efficiency, we build our multiplication gate in a hierarchical way, as shown in PTP, leading to untrusted interactions. However, we do not need to open commitments after every multiplication gate. We can open commitments forwardly only when it fails to open the final commitments.As with the addition gate, we can perform the same computation on the blinding factor PTP detects the attack by checking the commitments.Any tamper behavior during the decryption phase would never succeed because (4)Protocol Outputn ciphertexts, i-th of which is encrypted by The output is The input values are secretly shared by the input gate and the computations of the addition gate and multiplication gate are based on encrypted shares, which reveal no information about input values. According to the analysis above, the corresponding commitments of the output values can be opened and checked, ensuring the result is corrected.Participants require all the decrypted shares to recover the final result:a, b and c are random finite field elements unknown to everyone. Our off-chain protocol is similar to that in SPDZ [The off-chain preprocessing phase generates multiplicative  in SPDZ , but the in SPDZ ,35, genea, b and c are unknown to everyone and d equals 1. i-th share of Participants distributedly generate a quadruple Each participant Each participant All participants setAll participants compute Participants setParticipants invoke “Sacrifice” to check that indeed “Reshare” and “Sacrifice” are the same as that in SPDZ, and an overview of them is provided below:Protocol Reshare. Clients send transaction proposals to one or more peers with setting the endorsement policy. The endorsing peers then execute the chaincode to decide whether the transaction should be endorsed. If so, endorsing peers change the state on the ledger according to the targeted chaincode. An endorsement policy is a condition on what endorses a transaction . Thus a A critical detail is that all endorsing peers see identical transaction proposals (no matter whether they would be accepted in the next phase). Transactions are executed and verified in the endorsement phase so we run our on-chain compute protocol during that phase.PTP). As inputs are encrypted by different public keys, we need to deal with the question of how to perform decryption while executing multiplication gate. Unlike the solution of Benhamouda [A participant is a data provider who connects to the blockchain network by a client. Any participant can own at least one peer, through which he can join in one or more organizations in Fabric. As mentioned earlier, participants store encrypted inputs in the ledger  are dynamically written into the ledger as transaction proposals by clients in advance. The solution requires no code modifications to Hyperledger Fabric.To solve the second problem, we need some definitions to the data packet of the decryption request:mpcId is a unique id used to distinguish different computation requests. The stepId, which increases from 0, represents the progress of the current computation and is controlled by the chaincode. With mpcId and stepId, decryptors are able to check whether the request corresponds to a correct step by calculating the commitments locally and opening commitments after decrypting the data. If succeeding in opening commitments, decryptors store them into the cache and respond. If it fails, they ignore the request. Note that the results of decryption are some random values, which reveal no information about input values.The In this section, we discuss the security issue of our solution. As we pointed out at the beginning, the most critical problem is the leakage of the input data. No one should be able to learn anything except for their own inputs and outputs.Data privacy and Privacy control: The combination of homomorphic encryption and secret sharing allows computations to be performed on the encrypted shares. t and s) generated by decryptors are meaninglessly random values if no participant knows a or b of the i-th share of a and b, and no one knows a or b. Commitment x and a that would generate the same Comm. Even with the same private data a, x exist. If x is truly random, an attacker would be completely unable to figure out a [re out a .Resistance to collusion attacks: In our solution, a secret is broken into shares by the additive secret sharing scheme and then different shares are encrypted by different participants’ public key. The threshold of the additive secret sharing scheme is n, which means our scheme remains safe even there are up to PTP automatically verifies the data decrypted by decryptors by opening commitments so that decryptors fail upon tampering.Publicly verifiable MPC: Participants can verify whether the input value is valid and whether the dealer is honest by opening commitments. Due to commitments being public and automatically computed by PTP, everyone can check the correctness of the result by opening commitments. Although there are interactions in the multiplication gate, an active attack can be defended against, since any tamper behavior during the decryption phase would never succeed because PTP detects the attack by verifying the commitments.Trust model: The trust model of the proposed on-chain secure MPC protocol depends on the endorsement policy set when installing the chaincode. For example, if the trust model allows no more than k adversarial participants, we can simply set an endorsement policy that demands at least Our running environment was ubuntu 18.04, Intel(R) Core(TM) i5-6400 CPU @ 2.70 GHz, 16 GB of RAM and Hyperledger Fabric v1.4.0. We used the customized Docker container fabric-javaenv, as mentioned in In this experiment, we studied the impact of key size in Paillier Cryptosystem on running time. We jointly computed the weighted average of inputs from 10 participants and set i as the weight of (1) It can be seen that the total response time ranges from 91.61 milliseconds for 512 bits to 430.48 milliseconds for 4096 bits, and the CPU time in a single node ranges from 1.71 milliseconds for 512 bits to 14.3 milliseconds for 4096 bits. Both response time and CPU time grow with the increase in key size. This is normal because of the larger ciphertext and the increasing computational complexity, but it leads to higher security.(2) In addition, the CPU time is negligible compared to the total response time. Even when the key size is 4096 bits, the CPU time accounts for less than 4% of the total response time, and most of the time is spent on communication and reaching consensus, meaning that the performance has a lot of room to grow with the increasing optimization of communication channels.The NIST  recommenIn this experiment, we compared the response times for securely computing the sum of two inputs with an increase in the number of participants (computing nodes) based on three other schemes and our solution in this paper. The results are shown in Scheme 1 offloads private and intensive computations to an off-chain network. Similar to our solution, scheme 2 runs the secure MPC protocol on-chain and uses the Paillier cryptosystem. Scheme 3 has the same architecture as that in scheme 2, but we replace its homomorphic encryption scheme with BFV  to suppo(1) As illustrated, when there are no more than 20 computing nodes, our solution takes less time compared to scheme 1, but it seems that scheme 1 has a better performance when the network is large. This is because scheme 1 only stores MACs  and comm(2) We can see that scheme 2 consumes the least time. However, compared with our solution, the time consumption difference is quite small. Both of us use the Paillier cryptosystem, but organizations in scheme 2 encrypt values using the same key. Although they use access control list (ACL) rules to control access to the ledger, there still exists a conspiracy between the key owner and smart contract invoker. Differently, our scheme remains safe even there are up to (3) (4) In addition, we can see that, even for 20 nodes, the running time of our solution is about 350 milliseconds, which is longer than the time it takes to commit a block .In this paper, we proposed a publicly verifiable, secure MPC protocol consisting of two parts: an on-chain computation phase and an off-chain preprocessing phase. The scheme has the following advantages: (1) Privacy confidentiality: all input values and output values are in encrypted form and everyone learns nothing except his own input secret and output values. (2) Correctness and verifiability: Computations can be publicly executed and are controlled by no one. All the steps during computation can be publicly verified. We also described how the proposed secure MPC protocol can be integrated into Hyperledger Fabric, which helps to handle transactions that rely on confidential data owned by different participants. The scheme greatly guarantees the privacy of smart contract execution. The experiments showed that our solution had good execution efficiency. Meanwhile, most of the time taken to complete the protocol was spent on communication so the performance has a great deal of room to grow.The expensive communication costs limit our solution’s scalability to a larger network. In the future, we will explore the ways in which communication times and data exchange during computation can be reduced, or try to build a more efficient P2P communication channel for the decryptor. Moreover, using the proposed method to solve practical problems such as statistical analysis on credit data with guaranteed privacy is also among our plans.


Within complex tissue environments, such as the gastrointestinal tract and lung, these systems act to orchestrate and temporally align immune responses, regulate cellular function, and ensure tissue homeostasis and protective immunity. Group 3 Innate Lymphoid Cells (ILC3s) are key sentinels of barrier tissue homeostasis and critical regulators of host-commensal mutualism—and respond rapidly to damage, inflammation and infection to restore tissue health. Recent findings place ILC3s as strategic integrators of environmental signals. As a consequence, ILC3s are ideally positioned to detect perturbations in cues derived from the environment—such as the diet and microbiota—as well as signals produced by the host nervous, endocrine and circadian systems. Together these cues act in concert to induce ILC3 effector function, and form critical sensory circuits that continually function to reinforce tissue homeostasis. In this review we will take a holistic, organismal view of ILC3 biology and explore the tissue sensory circuits that regulate ILC3 function and align ILC3 responses with changes within the intestinal environment.Recent years have seen a revolution in our understanding of how cells of the immune system are modulated and regulated not only via complex interactions with other immune cells, but also through a range of potent  Innate lymphoid Cells (ILCs) are a family of innate immune effectors that localize mainly to mucosal surfaces and which play critical roles in regulating tissue immunity and homeostasis. The ILC family can be divided into three main subsets—group 1 ILC (ILC1), ILC2, and ILC3 based on their expression of master transcription factors and associated effector cytokine profiles [Reviewed extensively elsewhere ]. In thiILC3s are characterized by the expression of the retinoid-related orphan receptor γt (RORγt) , 5, 6 aninputs  are sensed and interpreted by ILC3 and give rise to functional outputs that culminate in the downstream modulation of tissue physiology to maintain health and homeostasis. While the inputs of these sensory circuits vary, and will be discussed in detail below, a major common ILC3-associated output is the secretion of effector cytokines including IL-22, IL-17A, IL-17F, and GM-CSF and lymphotoxin (LT)  , 7, 8 (Fxin (LT) . These soutputs). IL-22 signaling induces the production of antibacterial peptides such as RegIIIβ and RegIIIγ and S100 family members, which in turn regulate the commensal microbiota and limit access to the epithelial and mucosal niche (outputs) .Mammals have evolved multiple complimentary immunological mechanisms to promote the anatomical containment of commensal bacteria. These mechanisms enforce tolerance, suppress inflammation and maximize mutualism with the microbiota, and ILC3s have key roles in this process –15. ILC3al niche , 17. IL-al niche . Moreoveal niche : outputsoutputs) –21. In aoutputs) . IL-22 aoutputs) . In lineoutputs) , 28. Morractions : outputsCandida albicans . Indeed, ILC3-derived IL-17A and IL-17F are increased during intestinal inflammation in both mice and humans , 41. Toginputs). Indeed, early studies suggested microbial colonization of the neonatal intestine regulates the composition and size of the ILC3 pool within the intestinal tract. Pups born to germ free mothers were reported to have reduced frequencies of ILC3s—indicating a role of microbial signals in promoting tissue seeding by ILC3 subsets  (Gpr43 (Ffar2) in the modulation of intestinal ILC3 responses (inputs) —including butyrate, acetate and propionate—critical regulators of immune responses which are metabolized from dietary fiber by commensal microbes : inputs inputs) , 43. Levinputs) . SCFA cainputs) . Interesesponses : inputs (inputs) . Trigger(inputs) .inputs). As highlighted above, the availability and liberation of many dietary metabolites is also determined in part by mutualistic, commensal microbes within the intestine—while conversely the diet itself can modulate microbial composition and thus, determine the nature of host-commensal interactions. For example, the feeding of high fat diet (HFD) to pregnant mice was found to modify the expansion of ILC3 in the intestines of progeny through the modification of the mothers microbiota . Indeed, ILC3 are highly AhR dependent, and present severe functional impairments in the absence of cell-intrinsic AhR expression  ligands are normally liberated from cruciferous vegetables in the diet—such as broccoli and cabbage, but they can also be microbially derived . AhR is pression , 49–51. odentium . Intriguodentium , suggestIn utero exposure to the Vitamin A metabolite retinoic acid (RA) impacts directly on secondary lymphoid organ development with long-term immunological consequences (inputs). Moreover, it was shown that RA directly regulates the master transcription factor of ILC3, RORγt, and in the absence of maternal retinoids ILC3 failed to develop correctly . ILC3 numbers in the small intestine of mice deficient for the vitamin D receptor (VDR—KO mice) were shown to be increased, as was IL-22 expression, resulting in enhanced resistance to infection with Citrobacter rodentium (inputs)  . Vitamin(inputs) . In contfunction . Togetheinputs). In particular, CX3CR1+ intestinal MNPs cluster with ILC3 in distinct, organized lymphoid structures, such as CPs  (+ MNPs resulted in impaired IL-22 production by ILC3 and failure to control Citrobacter rodentium infection (+ MNP-derived TL1A further acts to augment IL-22 production from ILC3 (inputs)  act as key intermediaries and signal to ILC3 via the release of cytokine mediators during both homeostatic and protective immune responses , 61. Indh as CPs , 63. Micecretion : inputs nfection , 64, 65.rom ILC3 : inputs (inputs) .inputs to regulate ILC3 function. In particular, the central and enteric nervous systems are rapidly being appreciated as critical sensory and immunoregulatory systems.While microbial sensing by intestinal MNP and conserved crosstalk with ILC3 appear to be a major sensory circuit of intestinal immunity, emerging evidence suggests diverse sensory mechanisms across multiple biological systems provide additional It has been suggested that the immune and nervous systems are evolutionary linked, since they share functional similarities , 67. Bot+ cells present in the anlagen initiates a cascade of immune cell recruitment, in particular of fetal ILC3s, through sensing of neurotrophic factors that drive the formation of primordial lymphoid clusters (inputs and respond during both development as well as in the adult intestine (inputs). As mentioned previously, fetal and adult ILC3 development and function relies on RA signaling (Neuroimmune interactions are evident very early in life—and during the embryonic period the development of the enteric nervous system (ENS) and SLO organogenesis share many parallels. Notably, the neurotrophic factor receptor RET is essential for the development of Peyer's patches (PP) and also the ENS , 69. Morclusters , 69. Morntestine : inputs.ignaling . Intriguignaling , surprisMyd88-dependent manner; thus, implicating the enteric nervous system as a key player in environmental sensing circuits. In response to these cues glial cells secrete neurotrophic factors, which directly act on adult ILC3 cytokine production via cell-intrinsic RET expression (inputs). Ablation of Ret in ILC3s led to a reduction in IL-22, consequently impairing epithelial function and host defense to enteric bacterial infection   release by enteric neurons was also shown to regulate ILC3-derived IL-22 production through signaling via VIPR2, triggered in part by feeding and dietary cues : inputs inputs) , 75 (disinputs) –82. ThesIn addition to local environmental cues, mammals are also constantly exposed to a range of external stimuli and pressures such as fluctuations in temperature, oxygen levels and the daily light cycle. As a result many organisms have evolved circadian rhythms to align core biological processes with time of day, which are imprinted by an internal biological clock. Specifically, circadian rhythms are driven by cell-autonomous transcriptional feedback loops (“clocks”), which enable organisms to anticipate and adapt to temporal changes in their environment  and regulate metabolically demanding biological processes including body temperature, locomotor activity, endocrine responses, and feeding behavior—while on the cellular level circadian clocks regulate cellular metabolism and cell cycle , 84. In Per) and Cryptochrome (Cry), which in time translocate back into the nucleus and inhibit their own expression (Arntl (encoding BMAL1)  of the brain . The SCNpression , 87, 88.g BMAL1) , 87, 88.L1)   in a cell-autonomous fashion  resulted in altered ILC3 subset development—with mice exhibiting a marked reduction NCR+ ILC3s, while LTi-like ILC3 were unperturbed . Time-specific feeding altered daily circadian rhythms in clock related genes , 75inputcrobiota , 96.inputs)  are also known to be central in aligning many biological processes and in imprinting circadian rhythms. Indeed, signals from the central clock were shown to be a key regulator of ILC3 rhythmicity : inputs inputs) . Utilizial tract . The mecal tract . While iin vitro . Togetheinputs derived from third party cells stimulate outputs in ILC3, during both embryogenesis and adult life ILC3 provide the input and stimulatory cues to stromal cells to initiate a cascade of events that lead to the formation of secondary and tertiary lymphoid tissues.Unlike cells of the adaptive immune system, ILC3 are one of the first immune cells to colonize the intestine during the embryonic period and are critical for the formation of SLOs . In thisbona fide fetal lymphoid tissue inducer cells , which cluster with the LTo forming the primitive anlagen of the SLO  that start to express chemokines such as CXCL13, CCL19, CCL21, as well as the adhesion molecules VCAM-1, ICAM-1, and MadCAM-1 , 102. Th the SLO . Fetal I the SLO , 104. Co the SLO . This st the SLO . In addi the SLO . Therefo the SLO . Thus, aPostnatally, a large number of organized lymphoid structures designated as tertiary lymphoid structures start to form under the influence of environmental stimuli. These immune cell clusters include cryptopatches (CP), which are confined to bottom of the crypts within the intestinal lamina propria. CP formation is driven through similar molecular mechanisms to SLO, including via interactions between ILC3-associated LTα1β2 with the LTβR expressed by stromal cells and IL-7 signaling , 108. CPA unique feature of ILF development in comparison to LN, PP, and CPs is the requirement for microbial exposure. Intestinal bacteria are sensed by myeloid cells which increase the interactions between ILC3s and LTos, also via a LTα1β2 dependent axis, leading to increased expression of adhesion molecules by the stroma and recruitment of B cells to these structures , 114. ILCh25h, which acts to generate the cholesterol metabolite 7,α25-OH—a key migratory ligand for multiple immune cells including ILC3 (Gpr183), and facilitates localization of not only ILC3 but also T follicular helper cells, DCs and B cells to the follicular border of lymph nodes . Within the intestine-draining mesenteric lymph node multiple distinct stromal populations have been identified with differential capacities to attract immune populations and orchestrate immune cell crosstalk . One sucing ILC3 –120. Thiph nodes , 120–125t manner . TogetheILC3s are also emerging as key orchestrators and regulators of adaptive immune responses [Reviewed in detail in ]. This r3 and surface bound LTα1β2 interactions with DCs (outputs)  .outputs)  from epithelial cells : outputsoutputs) . Interesoutputs) . Conversoutputs) , 133, suinputs/outputs) (inputs/outputs) (outputs)  and reinforcing immune tolerance (outputs) . Similaroutputs) . Conversoutputs) : outputsoutputs) , implicainputs/outputs) (+ ILC3s were found to suppress effector CD4+ T cell responses toward the microbiota in the intestine (outputs)  on ILC3  NKT cell populations, and promote their functionality  molecules at levels comparable with other professional antigen-presenting cells and can acquire, process and present antigens (outputs) . Howeveroutputs) . In contntestine –139. In ntestine : outputsoutputs) . While t on ILC3 . In addiionality .inputs/outputs)  , 143. Reoutputs) . In addioutputs) , 146. Asoutputs. Thus, despite recent advances, one future challenge will be to understand how ILC3 integrate multiple concurrent signals from varying biological systems within a given tissue niche, and to determine how these cues are translated into cell fate decisions to determine the magnitude or quality of an ILC3 response. Many signaling pathways downstream of both cytokine and neuropeptide receptors converge upon core regulators of cell function—such as the mammalian target of rapamycin (mTOR)  . Moreoven (mTOR) . In linen (mTOR) . Engageminputs and outputs—and importantly the mechanisms through which these multiple sensory circuits are integrated and interpreted—will allow for new approaches to target this mucosal immune sentinel in the context of health and disease. Indeed, while ILC3 mediate many protective processes at homeostasis, dysregulated ILC3 responses have been implicated in a wide range of chronic inflammatory and metabolic diseases and have increasingly been suggested to play roles in cancer development and progression. Most notably, disruption of ILC3 responses is associated with the pathogenesis of inflammatory bowel disease (IBD)  , 150. Inse (IBD) . Thus, wse (IBD) , 95–97, In conclusion, ILC3 are strategically positioned within mucosal sites where they act as a hub of multiple distinct, yet complementary, sensory circuits. Together, these circuits act to continually survey the intestinal tract for perturbations in microbial, dietary and external environmental cues and enable the rapid communication and translation of this information, resulting in protective effector responses that continually reinforce normal tissue function and health. Strategies aimed at exploiting these cues and sensory circuits to promote or restore homeostatic ILC3 function, while simultaneously suppressing the dysregulated signaling associated with maladapted immune function, may lead to novel therapeutic intervention strategies in a number of human diseases.MH and RD conceived of and contributed to the writing of the manuscript. RD constructed the figures.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.


Hyperuricemia, or elevated serum urate, causes urate kidney stones and gout and also increases the incidence of many other conditions including renal disease, cardiovascular disease, and metabolic syndrome. As we gain mechanistic insight into how urate contributes to human disease, a clear sex difference has emerged in the physiological regulation of urate homeostasis. This review summarizes our current understanding of urate as a disease risk factor and how being of the female sex appears protective. Further, we review the mechanisms of renal handling of urate and the significant contributions from powerful genome-wide association studies of serum urate. We also explore the role of sex in the regulation of specific renal urate transporters and the power of new animal models of hyperuricemia to inform on the role of sex and hyperuricemia in disease pathogenesis. Finally, we advocate the use of sex differences in urate handling as a potent tool in gaining a further understanding of physiological regulation of urate homeostasis and for presenting new avenues for treating the constellation of urate related pathologies. One sucgyptians . Interesgyptians ,6,7 , which increases the risk of the precipitation of weakly soluble UA. Increased UA leads to precipitation of monosodium UA crystals, which can cause UA kidney stones and gout. The number of affected individuals continues to climb with current estimates being approximately 47.2 million HUA individuals in the United States; roughly 27.9 million individuals have severe HUA (>7mg/dL), with men being affected five times more often than women mg/dL, wh. HoweverHUA is just one of many conditions that have observable sex differences, as differing pathologies based on sex have been observed in a variety of fields ,15,16,17Although the overall kidney function seems to be improved in females when compared to males, once female kidney function does begin to decline, the resulting pathologies can be more extreme than in males, in part due to the fact that this decline is likely to occur later in life in women. As kidney function declines, SUA levels tend to increase, and increases have been reported to be associated with all-cause and cardiovascular mortality in a dose-dependent manner. Thus, as UA levels increase, so does the risk of developing other conditions, including CKD, hypertension, and diabetes mellitus ,53,54,55Taken together, this evidence implies that both biological sex and SUA level affect the kidney, in that the interaction of these two variables may influence renal function. The female sex must somehow affect the balance of UA levels, leading to a decrease in SUA when compared to males. Unraveling the mechanism behind this effect could lead to a better understanding of the underlying physiology and improved treatment for HUA. However, UA handling in the kidney is an incredibly complicated process, the details of which have yet to be fully elucidated.Renal UA handling involves multiple transporter proteins. UA is the protonated form of uric acid and enters the circulation as the terminal metabolite of purine metabolism in humans and the other great apes. It is produced from the degradation of purine nucleotides and amino acids, mediated by xanthine oxidase. Higher-order primates have lost the activity of the enzyme uricase, which further metabolizes UA to the much more soluble allantoin . Loss ofSLC22A6), OAT3 (encoded by SLC22A8), OAT4 (encoded by SLC22A11), and OAT10 (encoded by SLC22A13), as well as SLC2A9  [SLC17A1), and NPT4 (encoded by SLC17A3) are expressed [SLC22A12) and SLC2A9 have been reported [Of the UA excretion, 70% is mediated by the kidney and 30% by extrarenal pathways including the gut and the liver ,64,65. Ad GLUT9) ,72,73,74xpressed ,78,79,80reported ,81, resureported  and compFunctional data of these and other ,83,84 trSLC22A12 [SLC22A12 as potentially harboring the key gene. The resulting protein product, URAT1, proved to be a kidney-specific UA transporter, and variants were found in a Japanese family with significant hypouricemia [SLC22A12 variants causing hypouricemia have been reported at high frequencies in Japanese [ABCG2 [HNF1A and HNF4A [HNF4A variant by showing that it altered the ability of its protein product HNF4α to directly regulate trans-activation of ABCG2 [SUA levels display a strong heritable component, estimated between 40%–70% ,86,87. TSLC22A12 . A laboruricemia . SLC22A1Japanese , Korean Japanese , and EurJapanese  populatiJapanese , but theJapanese . ProgresJapanese ,86,87,92Japanese ,94. The Japanese ,101,102 e [ABCG2 , as wellSLC2A9, ABCG2, CAPN1, GCKR, IDH2, and SLC22A12 [SLC2A9 and ABCG2 were found to have significant associations in the more stringent genome-wide test for SNP effects on UA levels based on sex, confirming previously established findings [SLC2A9 had a stronger association with lower SUA levels in females, whereas ABCG2 had a stronger association with higher SUA levels in males, demonstrating that regulation and/or activity of these transporters may be influenced by factors that contribute to biological sex [SLC16A9, SLC17A1, SLC22A11, and SLC22A12), as well as UA transporter “keystone” scaffold protein encoded by PDZK1 [HNF4A and HNF4G [Only 6 of the 183 loci in that same study demonstrated sex-specific effect differences, including SLC22A12 . Howeverfindings ,103,104.ical sex . Signifiby PDZK1 . Additiond HNF4G ,106,107.ABCG2, SLC2A9, and SLC22A12. As mentioned above, variants in ABCG2 and SLC2A9 have been demonstrated to exert sex differences in effect size, but not SLC22A12. This may speak to one of the limitations of GWAS, such that these studies can only use common variants present in high enough frequencies to be detected, leaving rare but potential associated and causal variants to remain undetected in these studies. To address this, recent work that used whole-exome sequencing (WES) to probe for rare variants that contribute to SUA variance found numerous rare variants including two in the SLC22A12 gene (rs150255373 encoding R325W and rs147647315 encoding R434H) that had large effect sizes [SLC22A12 may have contributed to its under-reporting of sex effect. Additionally, SLC22A12 expression was found to be higher in males over the age of 50 compared to females of a similar age, yet SLC22A12 expression was the highest in females <50 as reported in a smaller transporter targeted study [SLC22A12 expression could indicate an increase in UA reabsorption, yet females under the age of 50 have some of the lowest levels of SUA of all populations sampled, implying that the reabsorption of UA by URAT1 may not be the primary contributor to SUA in females. This primary contribution is much more likely due to commiserate changes in other UA transporters. The picture of the genetic architecture emerging is one of two tiers, first a base composed of a large number of genes that provide a significant but small contribution to the variance in SUA, and then a top tier with the three critical UA transporter genes: ct sizes . The laced study . IncreasSLC2A9) and gout risk (ABCG2) for both adult [SLC2A9, including 90 rare variants that associated with SUA, and an additional number of common variants that map onto important functional regions of the protein [SLC2A9 are associated with both decreased SUA [SLC2A9 SNPs associated with a decrease in SUA, this decrease was much greater in women than in men , with a higher heritability component in females than in males in European populations [SLC2A9 contributed to a lowering of UA levels between 0.30–0.35 mg/dL or 5%–6% of all populations sampled, with a much stronger association in women [Not coincidentally, the two genes variants with the largest sex-specific effect differences are also the two with the common variants contributing the most to SUA variability  and HNF4A (encoding HNF4α) had significant associations with SUA [SLC22A12 [PDZK1 [SLC2A9 [ABCG2 [PDZK1 [HNF1A demonstrated a borderline significant increase in SUA and a decrease in FEUA in women, but not men [HNF1A and HNF4A may be influenced by sex hormones [HNF4A gene has been reported to have estrogen receptor (ER) enhancer elements [HNF4A locus between males and females [HNF4A has been further characterized in mice, in which male HNF4α-null mice demonstrated 1000 more affected genes than females [The effectors of UA homeostasis, the transporters, are an obvious mechanism for explaining sex differences but they exist downstream of other differentially regulated pathways, such as transcription factors. While several transcription factors have been implicated in the regulation of UA transporters, for example, HIF-1α and Nrf2 can regulate pression ,136,137,with SUA . Targetewith SUA . SpecifiSLC22A12  and impo2 [PDZK1 , while H [SLC2A9  and ABCG9 [ABCG2 , and als2 [PDZK1 ,142. Ind not men . Both HNhormones . The HNFelements , and theelements . In huma females , support females . Related females , demonst females . Thus, sTo explore the possible role of sex hormones in regulating UA transporters and more generally UA levels, an early study examined the effects of oral administration of synthetic sex hormones on both women and men . The stuABCG2 [ABCG2 can be transcriptionally regulated by estrogen. Additional reports have demonstrated that ABCG2 protein expression is down-regulated by estrogen [Estrogen has been reported to have direct effects on sex-associated UA transporters. For example, ER binding sites have been identified in the promoter region of ABCG2 , implyinestrogen , possiblestrogen . Based oestrogen . This stestrogen . DecreasUox gene, resulting in the production of the uricase enzyme, which then breaks down UA into allantoin. To remove this hurdle, a mouse with genetic ablation of the Uox gene was first described in 1994 [The complexity of human physiology and the heterogeneous nature of human populations make mechanistic studies difficult. Thus, effective model systems are needed to further progress our understanding of UA homeostasis and the role of sex in its regulation. Animal models are widely used to provide insights into human disease, but the specific differences in human and rodent UA physiology present additional hurdles ,155. One in 1994 , resultiSLC22A12 has been knocked-out on both a mixed [SLC22A12 knock-out models are grossly physiologically normal, with either an increased urinary UA to creatinine ration along with higher FEUA [SLC2A9 knock-out and there were no sex differences reported in the intestinal enterocyte-specific knock-out of SLC2A9 [SLC22A12 knock-out mice, SLC2A9 knock-out mice have limited use when exploring sex differences in HUA.Other key UA transporter genes have been targeted in attempts to raise the SUA levels in mice, including URAT1, SLC2A9, and ABCG2.  a mixed  and a pu a mixed . Both SLher FEUA  or increher FEUA . Whole-bher FEUA  demonstrf SLC2A9 . TherefoIn contrast, a new HUA mouse model produced by inserting a human ABCG2 variant , not only accurately recapitulates renal and gut UA transport defects observed in humans possessing the Q141K variant, but also results in profound sex differences in how the deleterious ABCG2 variant effects function. The mouse model was created in an effort to better understand if ABCG2 played a significant role in kidney UA excretion and the deleterious effects of the Q140K variant. The model demonstrated that ABCG2 is a key secretory transporter in the kidney and small intestines, however, while the Q140K variant was extremely deleterious in the intestines, it surprisingly had only a small effect on ABCG2 abundance and function in the kidney. This discrepancy in phenotypes appeared to explain why humans with the Q141K variant are at high risk for HUA: the mutant protein has little effect on transporter function in the kidney, thus differences in observed FEUA are small or are absent, yet the Q141K variant produces a profound loss of intestinal secretion of UA, driving increases in SUA .A second startling finding from the ABCG2-Q140K mouse model was that the female Q140K mice did not show any phenotypes . The femWith the right in vivo models, a number of important questions are possible to address. Since increases in SUA are associated with a myriad of other pathologies, including CKD, hypertension, and diabetes, it is difficult to determine which is causal and which is the co-morbidity. In the case of CKD, can HUA promote CKD, and will amelioration of SUA levels improve the prognosis for CKD? How does biological sex potentially contribute to this relationship? There is emerging evidence that treatment of SUA levels in patients with CKD can in fact improve CKD progression in patients receiving UA lowering therapy ,162. In Female sex hormones, specifically estrogen, may play a role in the regulation of expression or activity of UA transporters, specifically ABCG2 and SLC2A9. This is emphasized by the fact that females may have differences in renal UA transporter expression, localization, or activity. Estrogen could mediate either direct transcriptional regulation of the transporter genes, or activate transporter specific transcription factors, including HNF4α. Observable differences in how transporter transcription and post-translational modification are regulated between the sexes may reveal significant information about these regulatory pathways and provide targets for therapy. Thus, using sex as a biological variable may provide key insights into understanding UA handling, elucidating underlying physiological regulatory mechanisms, and underscoring a critical need for future studies.


Dear Sir,p = .009), while the G1, G3 and G4 groups have no correlation in Table 2. Multivariate Cox regression analysis showed that, using G2 as the reference, the all-cause mortality was significantly higher in G5 than in G2 for Models 1and 2, while no difference in other groups in Table 3. I pay special attention to the results of this research because it caused me some confusion. Therefore, we cite several studies to compare and contrast with the findings of Hiroyuki et al.We have carefully read the study published by Yoshida et al. on the extreme hyperuricemia is a risk factor for infection-related deaths in incident dialysis patients: a multicenter prospective cohort study . Uric acp = .008), as well as the HR for all-cause mortality among cases in the Q5(≥8.5 mg/dL) compared to the Q1(≤5.8 mg/dL) was 0.65 . All laboratory data were based on initial values entered in the Korean Society of Nephrology registry.Firstly, a retrospective cohort study conducted by Kim et al. . 7333 he2 = 16.11, p < .001). For each 1 mg/dL increase in baseline UA levels, the all-cause mortality HR was 0.55 . The all-cause mortality was significantly low in HD patients with serum UA levels of ≥6.2 mg/dL.Secondly, Beberashvili et al.. conductp < .05; HR 1.64, 95%CI 0.92–2.89, p < .05) compared to the reference group (7.0–7.5 mg/dL). In the adjusted model, both lower(<5mg/dL) and higher (≥8 mg/dL)) UA levels were independently associated with higher adjusted HR  of all-cause mortality compared to the reference group (7.0–7.5 mg/dL). Similar findings were reported by Chang et al. [p = .016; HR 4.69, 95%CI 1.24–17.72, p = .023). Both TA-UA <6 and ≥8 mg/dL increased the all-cause mortality in PD patients.Finally, An observational study by Sugano et al.. using aIn conclusion, I think the inconsistence of study results may partly be associated with the use of only baseline UA data. I suppose TA-UA is the best indicator to evaluate the UA levels in PD patients. Pre- and postdialysis UA difference may be the most appropriate reference for controlling UA in HD patients. Further prospective studies of larger sample size are needed to confirm this relationship and to clarify the underlying mechanisms such as the role of UA in antioxidant capacity.


This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP . A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min. Hencetis (HU) , arthroptis (HU) , and HTLtis (HU)  and HAM/tis (HU) ,10 have patients . Consequpatients ,13, whicpatients . It has patients .LabDisk cartridge for HTLV-1 testing integrates the complete workflow: insertion of a finger prick blood sample into the LabDisk, nucleic acid extraction with subsequent digital reverse transcription (RT-)LAMP (dLAMP), and fluorescence readout. The LabDisk cartridge enables automated liquid handling and contains pre-stored reagents for nucleic acid extraction, reverse transcription of RNA, and LAMP. This integrated functionality in combination with the benefits of absolute quantification by digital mediator displacement (MD) LAMP demonstrate the potential for the next innovation cycle of POC analysis and could, with appropriate development, replace state-of-the-art instruments such as the Alere™ q  for HIV-1/2, the SAMBA® , the Xpert VL  for HIV-1 and hepatitis B/C, or the EOSCAPE-HIV  for HIV-1.A significant challenge in the development of a POCT test for HTLV-1 is the integration of sample preparation from whole blood, due to the virus’s low abundance in the plasma of infected individuals . This is+ T-lymphocytes, designated as MT-2 cells [Genomic HTLV-1 nucleic acids were isolated from cell cultures (HTLV-1-infected CD4-2 cells ) with MaBst 2.0 WarmStart DNA Polymerase , 20 U Transcriptor Reverse Transcriptase , primers , 0.1 μM mediator, and 0.05 μM universal reporter . For the dLAMP, the reaction mix was incubated for one hour at 64 °C.The dLAMP assay includes the reverse transcription of viral RNA, the amplification of c-DNA and proviral DNA, and the sequence-specific detection of LAMP amplicons by mediator displacement (MD). The primers and the fluorescent universal reporter for the dLAMP are taken from  and listMicrofluidic actuation, incubation, and fluorescence readout were conducted in a functional model of a standalone POCT instrument . RotatioLabDisk cartridges were manufactured by soft lithography in the Hahn–Schickard prototyping facilities [LabDisk cartridges was achieved by microthermoforming 300 µm COC 6013/8007 foils  using a blow moulding process, which was implemented with a HEX01 hot embossing machine . After microthermoforming, all pre-stored reagents were deposited into the LabDisk cartridge. Pressure-sensitive adhesive foil 9795R  was used to seal the microfluidic cartridge. All the reagents, except the proteinase K, are pre-stored on the microfluidic cartridge as depicted in The microfluidic cilities . In brieLabDisk   that can collect up to 50 µL of whole blood. Thanks to the pre-storage concept, the user only has to insert the blood-filled capillary into the LabDisk a and inj see ESI  starts.LabDisk  the performance of fully automated sample-to-answer testing. The dLAMP assay performance with dried reagents, including pellets of lyophilized reagents, primers, MD oligonucleotides, and Tris, was investigated with manually extracted HTLV-1 nucleic acids using the microfluidic chip, and yielded results (7 ± 5 copies per MT-2 cell) comparable to the reference method with fresh reagents. The total number of copies per assay is plotted against the initial MT-2 cell number per assay in A reliable estimation of the average copy number of HTLV-1 nucleic acids per MT-2 cell appears difficult, because the process of HTLV-1 replication in vivo remains poorly understood. Reports in the literature are inconsistent, and values vary between a single proviral copy per MT-2 cell ,32 and mLabDisk was assessed by mixing 5 µL of MT-2 cells from fresh culture with 45 µL of whole blood to reach a total volume of 50 µL. The mix was collected in a capillary and transferred into the sample inlet chamber on the LabDisk  spiked with MT-2 cells from fresh culture (5 µL). According to reports in the literature [After verifying the functionality of rehydrated reagents and the efficiency of extraction on the terature , the numtax mRNA level, which correlates with disease progression. In HAM/TSP patients, an increased HTLV-1 tax mRNA level is accompanied by an increased HTLV-1 proviral load, which is also a marker for disease outbreak [tax mRNA levels, but high proviral loads. Consequently, in the latter case, it would be advantageous to differentiate between the mRNA level and the proviral load, for example, through selective amplification of DNA by omitting reverse transcription or by selective amplification of RNA [The POCT test presented herein detects the provirus (DNA) integrated into the genome of the human host cell as well as viral RNA. As HTLV-1 exists mainly as provirus, there is no detectable virus in the blood plasma or the host cells, and thus the detected RNA indicates the HTLV-1 outbreak . Howevern of RNA ,37.The processing time for the automated sample-to-answer testing was 2 h 10 min, including 60 min for nucleic acid extraction, 60 min for dLAMP, and 10 min for readout of the fluorescence signals. The presented system requires at least twice as long as currently available systems, such as the Alere™ q for the detection of HIV-1/2, which requires approximately 55 min processing time. The potential to reduce the processing time lies in the adjustment of the dLAMP reagent composition and the extraction steps.LabDisk platform. The automation and miniaturization of the laboratory workflow, including nucleic acid extraction, reverse transcription, and digital amplification with integrated fluorescence readout, reduces manual handling steps to the insertion of the blood capillary and the proteinase solution, thereby providing a quantitative HTLV-1 test for the point of care. Precise quantification is achieved by digital mediator displacement reverse transcription LAMP (MD RT-LAMP), removing any need for standard curves. The quantified HTLV-1 proviral load in blood samples spiked with infected MT-2 cells was comparable to a manual reference test (7 ± 3 vs. 7 ± 2 nucleic acid copies per MT-2 cell). The results were available after two hours and 10 min. Our novel system stands out because of its ability to provide calibration-free, digital sample-to-answer quantification at the point of care, as well as for its potential to be adapted for the implementation of different microfluidic applications.In the present work, we demonstrated a POCT system for fully automated, digital HTLV-1 proviral load quantification from whole blood based on a centrifugal microfluidic Further steps towards the development of a commercial product should include the pre-storage of proteinase K in the inlet chamber or in the blood capillary in dried form. In addition, the time to result should be reduced to <60 min. This can be achieved by optimizing the dLAMP, microfluidic workflow, or POCT instrument. A significant reduction of the incubation time of the dLAMP to less than 25 min can be achieved by adjusting the enzyme concentration, titrating primer concentrations, or using enhancing LAMP additives. Another approach deals with the time reduction of single steps in the microfluidic workflow. For instance, all bead handling holding times were kept as defined by the manufacturer in the manual reference workflow. However, efficient mixing within the microfluidic structure could decrease the binding, washing, and elution times and result in a lower total time. Heating steps by the POCT instrument were quite time-consuming and an improvement of the heating system could decrease the total workflow time. However, these steps were not part of this work. Furthermore, the expansion of the assay to additional targets could allow the detection of co-infections, e.g., HTLV-1/HIV.


Under the one-child policy of birth control, total fertility rates (TFRs) declined rapidly among women in China. TFRs dropped from 2.29 in 1990 to 1.18 in 2010 and to 1.05 in 2015. However, little is known about the evolution of fertility patterns in China during 1990–2015.We used population data from 1990 to 2015 and applied age–period–cohort (APC) models to examine temporal changes and used regression models to analyze the effect of education on fertility across periods and cohorts in China.Age effects assume an inverted U-shaped curve, which increase and then decline across ages, with a peak value in age groups 20–24 or 25–29. Period effects show a U-shaped curve, which first decline and then increase. Cohort effects show an inverted U-shaped plus V-shaped curve, which first increase, then decline and rebound with different age effects and period effects. The APC effect curves of all-order births are similar to those of first birth, but with different magnitudes.We revealed the evolutionary trends in fertility patterns among Chinese women from 1990 to 2015. The one-child policy exerted a crowding out effect on education. Even if the well-educated women had an intense fertility intention, the fertility policy offset their desire for more children. Women’s fertility rapidly declined under the constraints of the one-child policy in China. The period 1990–2015 is a phase of sustainably low fertility and also affects future demographic trends. However, little is known about the evolution of fertility patterns in China that are attributable to the impacts of age, period, and cohort. In this study, we established models to analyze the evolutionary progress and underlying causal relationships during 1990–2015. Results show temporal changes in fertility over a period of 25 years. Fertility patterns in urban regions are clearly different from those in rural areas. The effect curves of first birth are similar to those of the all-order births, but with different magnitudes, showing remarkable differences not only with respect to age but also period and cohort. The influence of education on fertility is not well reflected under the one-child policy and moreover, the rigid fertility policy exerted a crowding out effect on education. With implementation of the universal two-child policy, the crowding out effect of the fertility policy on education is weakened. Incentives to support this policy should be introduced, to help resolve the issue of high childcare costs. We wish that the birth quota would be abolished in the near future, to help China address the problem of rapid population aging.The period 1990–2015 is special in China’s fertility history, representing a sustained era of low fertility. Under the constraints of the one-child policy in China, women’s total fertility rates (TFRs) rapidly declined to below the replacement level (2.1). TFRs dropped from 2.29 in 1990 to 1.18 in 2010 and to 1.05 in 2015. Population aging, shrinking of the labor force, low fertility, and related socioeconomic problems forced the Chinese government to take action. The trend of persistently low fertility forced China to relax its birth control and implemented a universal two-child policy at the end of 2015, allowing nearly all couples to have two children. Still, fertility continues to decline. We wonder if China formed a unique fertility pattern with far-reaching effects during the period 1990–2015, when TFR declined from a level a little below 2.1 to the lowest fertility rates ever recorded. The aim of the paper is to review the historical trend of China’s low fertility and shed light on the formation of this pattern. We used the population data from 1990 to 2015 and applied age–period–cohort (APC) models to examine temporal changes and used regression models to analyze the effect of education on fertility across periods and cohorts in China. The evolutionary trends of Chinese women’s fertility patterns should be considered not only as related to China’s economic, cultural, and policy factors but also within the Chinese historical context on how fertility patterns evolved and what has driven those patterns in the country.Both fertility level and desired fertility have trended downward since the 1980s, together with increased economic and educational development in China. Moreover, fertility levels in the country have been lower than desired fertility since 1990 . Overalli(t)* denotes the adjusted TFRi(t), and ri(t) represents the rate of change from the beginning to the end of year t, in the mean age of childbearing at birth order i. Compared with the conventional TFR, the adjusted TFR was considered more helpful to assess fertility trends. Although the Bongaarts–Feeney adjustment is attractively simple, there exist some shortcomings such as unclear conceptual foundation, assumptions that rarely characterize actual populations [The evolution of fertility patterns is associated with timing factors. Fertility timing effects are often described as period, cohort, or other temporal variables, e.g., tempo. However, tempo distortions would occur in the calculation of the TFR. Therefore, Bongaarts and Feeney  establisulations . It doesulations , even maulations . In factAge effects represent the variations associated with different age groups that are mainly brought about by such events as physical and physiological changes and aging, and that are closely connected to reproductive conditions. Period and cohort effects reflect the influences of social and environmental forces. Period effects represent variations often resulting from shifts in social, cultural, economic, or physical environments or historical events, which influence all age groups, for instance the adjustment of fertility policy. Cohort effects represent variations in fertility across groups of women born in the same year(s) who shared common social background and experienced common historical events, reflecting the effects of early life exposure to socioeconomic, behavioral, and environmental factors that act persistently over time to produce differences in life course outcomes , 8, 9.An important pattern is that women who give birth earlier have more children than those who start late , 11. TheThe cohort perspective on fertility postponement can be traced back to Ryder’s research , which fChina has a large population and has undergone a huge demographic transition within such a short time. This means that the period effects and cohort effects would be much more different than those of Western countries. Therefore, according to effect analysis evolutionary trends in fertility could be explained. However, little is known about the evolution of fertility patterns in China that are attributable to the impacts of age, period, and cohort. Owing to identification problem, few demographic studies have succeeded in separately estimating the contributions of age, period, and cohort variation during the evolution of fertility patterns. Few studies have been done to summarize Chinese women’s fertility trends in recent decades, and analysis is lacking of the underlying historical events, which would have an influence on this progress. In this study, period effects, age effects, and cohort effects are analyzed to distinctly uncover the evolutionary trends in Chinese fertility patterns. Within a few decades, China’s fertility policy has stabilized  and womeThe evolution of fertility patterns is the result of many factors or important events. Education is an important proxy indicator to measure development, also employed in much demographic research , 20. TheThe questions arise of which is the greater driving force, the one-child policy or educational expansion, and how do these interact with each other? In this study, we took childbearing-age women as the research sample and used APC models to analyze the evolutionary progress and underlying causal relationships during 1990–2015. We established models to estimate the influence of educational improvement, on the basis of age-, period-, and cohort-effects analysis. Under the constraint of a stable fertility policy, we examined how improvement in women’s educational attainment affected their fertility. We aimed to reveal the temporal patterns embedded within the declining fertility and provide evidence to help researchers and policymakers understand the trajectory shaped by temporal effects.APC models were initially introduced by Mason et al.  as a genF is the age-specific fertility rate (ASFR), a is a constant term, A is the effect of age group, P is the effect of the survey period, C is the effect of the birth cohort, and ε is the disturbance term.The basic APC model is written as follows:We used a Poisson log-linear model to analyze the evolutionary trends in Chinese fertility patterns.F is assumed to be distributed as a Poisson variate. i.e., i  in the jth year .i and the average number of women in age group i in the jth year.ith age group,jth year, and kth cohort. One of each of three coefficients is set to zero as the reference age, period, or cohort category against which the estimated coefficients for other categories can be compared [Fertility compared . ParametFertility is the ASFR (‰). The independent variables are as follows: Age is the age of childbearing women and its squared term (Age squared) is mainly used to capture the possible nonlinear effects of age on fertility. Educational attainment qualifications for Edu were disaggregated into seven levels: illiterate (less than a primary school education completed), primary school completed, junior high school completed, senior high school completed, junior college or post-secondary certificate completed, undergraduate university education completed, and graduate-level education completed. Edu was measured using average years of education  in the statistical years. Period is the period dummy . a is the intercept. Zone is the dummy for region, including cities, towns, and villages . α, β, χ, and δ are coefficient terms, and ε is the error term.We established regression models to estimate educational effects on the evolution of fertility patterns, controlling for the variables age, policy, period, and region. and Lee ) and setData on fertility used in this study are obtained from the National Population Censuses in 1990, 2000, and 2010, and the 1% National Population Sample Surveys in 1995, 2005, and 2015 in China. China has experienced a long period of low fertility since the 1990s. During that period 1990–2015, the fertility policy  has been rigorously implemented, which ensures data continuity and helps in exploring the evolution of fertility patterns among Chinese women. In the following analysis, we disaggregated women of childbearing age (aged 15–64 years old) into seven 5-year age groups: 15–19, 20–24, 25–29, 30–34, 35–39, 40–44, and 45–49 years. 764,717,850 individuals were involved in sampling which were grouped into 168 observation data. We used six periods and 12 successive, 5-year birth cohorts labelled according to their central birth years, to analyze fertility behaviors. To precisely investigate rural–urban variations, we divided the regions into four types: the whole country/national, cities/urban, villages/rural, and towns. We reported the results separately.Figure Figure The estimation results of the APC models using the IE method for fertility are reported separately according to all-order births and first birth in the different regions Table . We firsAge effects assume an inverted U-shaped curve. In Fig. Period effects show a U-shaped curve. The size of period effects first decrease and then increase. This has been negative since 1995 and has continuously dropped to the minimum and then rebounded (it is positive in cities and towns in 2015). This indicates that period effects have contributed to the decline in fertility. If we set the period effect in 1990 to 1, it is 0.38 in 1995 and rises after 2000, from 0.28 in 2000 to 0.39 in 2015. This indicates although fertility policy isn’t relaxed in such a low-fertility situation, there exists a little more intensive fertility intentions. Actually, the problems caused by low fertility and an aging population have forced the birth control policy to be gradually relaxed, and the period effects tend to be stronger with relaxation of the fertility policy. In 2011, couples were allowed to have a second child if both partners were only child, then in 2013, couples were permitted to have a second child if one partner was only child. At the end of 2015, the universal two-child policy was fully implemented. The trends of period effects in cities, villages, and towns are the same, first rising and then falling. The period effects in cities were with a minimum of 0.24 in 2000, but rebounded most intensely. On the contrary, period effects in villages were less affected because fertility policies were loosely implemented. The period effects of first birth are lower, then similar to, then higher since 2005 than those of the national all-order births, which were higher in cities than those in villages during 2005–2015.As Fig. Age squared is negative, forming an inverted U-shaped curve. The empirical results accord with the reproductive facts, and there is a nonlinear relationship between fertility and age in which fertility increases with age, then reaches a peak value and declines. Model 3 shows that the inflection point is at ages 26–33 years, in accordance with the data analyzed in Table The results of regression models are displayed in Table Edu is negative at the 10% level. With controlling for periods and regions (Model 2), R2 increases and the coefficient of Edu improves to become positive at the 1% level. The inconsistency in the results of Model 1 and Model 2 shows that there may be missing variables. So, in Model 3, we further controlled for special background factors in China, i.e., the fertility policy Pol. We find that the increase of R2 and Edu is no longer statistically significant. This indicates that the effect of education on fertility has not been reflected correctly under the control of the one-child policy. The rigid one-child fertility policy exerted a crowding out effect on education. Even if well-educated women had intense fertility intention, the fertility policy offset their desire to have more children. This finding accords with the actual situation in China. The rigid one-child policy depresses the fertility desire that arises with improvement in SES, living standards, and educational attainment, which accords with the above-analyzed cohort effect. Model 3 indicates that the period effects are negative, which means that the period’s negative effect on fertility is enhanced.The results displayed in Table Age is a key factor affecting reproductive physical and physiological conditions, as are period and cohort, considering socioeconomic conditions and historical events in explaining fertility transitions. We analyzed the APC effects of fertility and explained the evolutionary trends in fertility among Chinese women and the characteristics of these trends, together with other factors, such as China’s one-child policy, the improved living conditions resulting from China’s Reform and Opening-up, and the increase in childrearing costs. On the one hand, the fertility policies are very different, from no control to family planning programs and up to the recent policy loosening. On the other hand, the socioeconomic status and educational conditions experienced among women in these cohorts are also remarkably different. Thus, the cohort effects of women born in different eras differ, with era characteristics. One possible reason for this is that women’s fertility intentions intensify with improvement of living conditions, which roughly fits Easterlin’s relative income hypothesis , 34. HowMoreover, we examined the effect of improvement in educational attainment among women on fertility transitions. APC effects show temporal changes in fertility over a period of 25 years. In fact, the period effects and cohort effects are related to the fertility policy and reflect the changes across different eras. Given that fertility policies pursue in-time fertility changes rather than economic and social changes, period effects would be much greater than cohort effects. With implementation of the universal two-child policy, the crowding out effect of the fertility policy on education is weakened. Nevertheless, childbearing costs  would suppress, even offset one’s desire for a second child . More efThe year 2015 represents a turning point in the fertility history of China. However, late childbearing is an irreversible trend in China. After implementation of the universal two-child policy, fertility desires were affected, especially at the beginning of this policy. Well-educated or/and older women of childbearing age became more willing to have a second child. Thus, later fertility could rebound greatly. Staggered maternity leave for women of childbearing age should be advocated according to older ages of childbearing. However, later childbearing also means higher reproductive health risks and costs for infant care. Preventing later childbearing or taking measures to encourage women to give birth at the younger ages  would help to relieve population aging and fertility declining. We advocate the adoption of financial policies to discourage further delays in childbearing. Health services could provide greater support for late-childbearing women to reduce reproductive risks and maintain a balance between family and work. Moreover, we wish that the birth quota would be abolished in the near future, to help China deal with the rapid population aging. Actually, China’s aging is really serious. According to UNDP’s projections, the proportion of population aged 65 and above in China would increase from 9.6% in 2015 to 27.6% in 2050, meanwhile the old-age dependency ratio would increase from 0.13 to 0.47 .Despite the above-mentioned findings, the study has several limitations. First, the existing statistical data may not be enough for analyzing fertility trends, and more sampling, more longitudinal/tracing data may enhance the persuasiveness of results. Second, due to the cross-sectional design of the study, the effect of education over time is not known, and how education operates during women’s childbearing span should be considered in order to gain more understanding about the relationship between education and fertility. Third, additional new models and methods are needed for testing the influence of historical events on the evolution of fertility trends. Last, the situation in the field of fertility is changing since implementation of China’s two-child policy. According to data of the National Statistical Bureau of China , the numThe period 1990–2015 is a critical phase in the demographic evolution of China and also affects future demographic trends. Based on descriptive analysis and APC accounting models, we provide a summary of temporal effects on fertility trends, which can be delineated into changes across ages, over periods, and among birth cohorts. We revealed the evolutionary trends of fertility patterns among Chinese women using APC analysis of fertility from 1990 to 2015, and found that the fertility patterns are clearly different between urban and rural regions.First, age effects assume an inverted U-shaped curve. The peak of age effects in cities is in age group 25–29 and not 20–24, compared with that in villages and towns, which means that the peak of fertility intention in cities emerged much later than in villages and towns.Second, period effects on fertility show a U-shaped curve. The size of period effects first declines and then rises. The trends of period effects in cities are not different from those in villages and towns, first increasing and then declining. The period effects in cities were with a minimum of 0.24 in 2000, but rebounded the most intensely.Third, cohort effects show an inverted U-shaped plus V-shaped curve. Cohort effects first rise, then drop, and rebound in the end, which are much more different with age effects and period effects. The effect curves of first birth are similar to those of the all-order births, but with different magnitudes, showing remarkable differences not only with respect to age but also period and cohort. The age effects of first birth are much more important in the “Golden Ages” (20–29 years). Period effects enhance, more remarkably in cities than in villages. Cohort effects become much more important after enforcement of the one-child policy.Last, the influence of education on fertility is not well reflected under the one-child policy. The rigid fertility policy exerted a crowding out effect on education. The analysis of women’s educational attainment shows that even though well-educated women had a strong fertility intention, the rigid birth-control policy offset their desire for more children.


Osteosarcoma is one of the most commonly seen bone malignancies with high incidence rate in both children and adults. Although the regulatory network of osteosarcoma has been greatly concerned for years, the mechanisms regarding its oncogenesis and development are still not clear. Recent discoveries have revealed that long noncoding RNAs (lncRNAs) play a crucial role in the development, progression, and invasion of osteosarcoma. Deregulated expression of lncRNAs has been found to participate in the regulation of various signaling transduction pathways in osteosarcoma. This review summarized roles of lncRNAs in the pathogenesis, development, and potential therapeutic of osteosarcoma via different signaling pathways. For examples, MALAT1, CCAT2, FER1L4, LOXL1‐AS1, OIP5‐AS1, PVT1, DBH‐AS1, and AWPPH regulate PI3K/Akt signaling; AWPPH and BE503655 regulate Wnt/β‐catenin signaling; NKILA and XIST regulate NF‐κB signaling; MEG3 and SNHG12 regulate Notch signaling; FOXD2‐AS1 and LINK‐A regulate HIF‐1α signaling; GClnc1 and HOTAIR regulate P53 signaling; ZFAS1, H19, and MALAT1 regulate MAPK, Hedgehog and Rac1/JNK signaling, respectively. Although initially considered to be transcriptional noise or so‐called “Junk” for lack of coding potential, numerous ncRNAs have been found to play pivotal roles in cellular processes of different diseases, including cancer.Multiple mechanisms have involved in the lncRNAs regulatory effects of gene expression. For instance, lncRNAs may act as epigenetic regulators, signals for transcription promotion, decoys for transcription repression, or scaffolds to form ribonucleoprotein complexes by interacting with proteins. LncRNAs can modulate the stability and translation of mRNAs, act as precursors of miRNAs, as well as regulating the distribution of miRNAs et al In general, lncRNAs have been found to regulate gene expression at transcriptional and/or post‐transcriptional levels.2Cell signaling plays a crucial role in cellular process and has a great impact on the modulation of gene expression in various cancers. Emerging evidence suggests that lncRNAs are concerned with OS through a variety of signaling pathways. For example, lncRNA HOTAIR promotes the proliferation and invasion of OS cells via the AKT/mTOR signaling pathway.2.1Numerous reports confirm the role of AKT whose activity usually involves various regulators in cancer. Among them, the most important one is PI3K, known as a positive Akt regulator. The PI3K/Akt signaling pathway plays important roles in different cellular processes, such as tumor cell growth, apoptosis, migration, and invasion.Colon cancer‐associated transcript 2 (CCAT2), located in the hotspot of the tumor‐related rs6983267 SNP, has been associated with increased predisposition for several cancers.The long noncoding RNA Fer‐1‐like protein 4 (FER1L4), initially identified in gastric cancer, is involved in tumorigenesis of numerous cancers such as hepatocellular carcinoma and glioma.Other lncRNAs, such as LOXL1 antisense RNA 1 (LOXL1‐AS1), OIP5‐AS1, PVT1, DBH‐AS1, and AWPPH, have been detected in various human cancers,2.2Wnt pathway mutations have been frequently observed in cancer. Aberrant activation of Wnt/β‐catenin signaling is closely related to various cancer types as it controls a number of oncogenes, which leads to cell proliferation and tumorigenesis.Numerous dysregulated lncRNAs have been identified by RNA‐seq, such as lncRNA BE503655, whose function and regulatory mechanism were studied.2.3Nuclear factor‐κB (NF‐κB), a widely expressed transcription factor, is one of the most significant molecules connecting inflammation with cancer. The activation of NF‐κB pathway is regulated by multiple mechanisms and plays a key role in cancer progression including cell growth, invasion, and metastasis.The lncRNA X‐inactive specific transcript (XIST) has been discovered to promote cancer development and was related to poor prognosis.2.4As a highly conserved cell signaling pathway, the Notch signaling is also essential to multiple biological processes and participates to the development of various cancers.Small nucleolar RNA host gene 12 (SNHG12), a newly discovered lncRNA, has recently been found upregulated in several types of tumors including OS.2.5As the most significant modulator in the response to hypoxia, HIF‐1α has been confirmed to regulate hypoxic gene expression via signaling transduction networks. Numerous studies have revealed the existence of lncRNAs in hypoxic tumor regions. One of them is FOXD2‐AS1, a novel lncRNA which may function as a miRNA sponge to modulate tumorigenesis. The aberrant expression of FOXD2‐AS1 has been found to correlate with HCC by silencing CDKN1B (p27) via recruiting EZH2, an important element of PRC2 (polycomb repressive complex 2 to its promoter region.LincRNA for kinase activation (LINK‐A) is an oncogene in breast cancer, in which it activates HIF1α to promote triple‐negative breast cancer progression.2.6As a tumor suppressor, P53 has been discovered mutated in more than 50% of malignancies. p53 regulation mainly refers to translation and posttranslational modifications as well as protein stability. Increasing evidence revealed that lncRNAs are involved in the p53 pathway with different mechanisms of action.HOTAIR was the first tumor‐related lncRNA discovered to be associated with cancer metastasis and poor prognosis. In a recent study concerning OS, HOTAIR was also detected to be upregulated as an oncogene which promotes proliferation and inhibits apoptosis in OS cells. Furthermore, the expression level of p53 after silencing HOTAIR is significantly upregulated, while bcl‐2 expression is downregulated, indicating that HOTAIR may take part in the p53‐mediated apoptosis pathway in OS cells.2.7Some signaling pathways are also correlated with lncRNAs in OS but are relatively rarely found. For instance, Zinc finger antisense 1 (ZFAS1), a newly identified lncRNA, was reported to be upregulated in OS cells and promote colony formation, migration, and invasion of OS cells via activating the MAPK signaling pathway.3Most lncRNAs do not seem to be as significant as molecules of signaling transduction on their own. However, they are crucial in regulating signaling pathways which tend to be more adaptable to tumor microenvironment. One of the most significant roles of lncRNA regarding cell signaling regulation in OS is that lncRNAs might act as a scaffold through which lncRNAs can directly interact with various signaling molecules.This assay was reviewed and approved by the Ethics Committee of Ningbo NO.6 Hospital. All participants signed written informed consent documents.