Chronic unpredictable mild stress (CUMS) mice were established, and depression-like behaviors were confirmed using the forced swimming test (FST), sucrose preference test (SPT), and the tail suspension test (TST). Next, the expression of miR-212 and its potential target, i.e., nuclear factor I-A (NFIA), was verified using quantitative reverse transcription (qRT)-PCR analysis and Western blotting in CUMS mice. The effects of miR-212 and NFIA on depression-like behaviors, inflammatory response, and neuronal apoptosis were examined using FST, TST, SPT, enzyme-linked immunosorbent assay (ELISA) assay, and flow cytometry analysis. Finally, the relationship between miR-212 and NFIA was examined using a dual-luciferase reporter assay. Based on our findings, miR-212 was significantly upregulated, while NFIA was downregulated in CUMS mice. miR-212 overexpression could suppress the CUMS-induced weight loss, immobility time in FST and TST, and increased hippocampal neuronal apoptosis and pro-inflammatory cytokines levels. In addition, NFIA upregulation could partially reverse the effects of miR-212 mimic in CUMS mice. Accordingly, miR-212 could ameliorate CUMS-induced depression-like behavior in mice by targeting NFIA, indicating its protective role in depression.Depression is characterized by persistent depressed mood and cognitive dysfunction, severely impacting human health. In the present study, we aimed to explore the role and mechanism of microRNA (miR)-212 in depression  Depression is one of the most common psychiatric disorders, primarily characterized by the patient’s depressed mood, loss of sense of enjoyment or interest in the outside world, self-perception of incompetence or inadequacy, depressed psychological needs, and disconnection from society . These mIn recent years, there have been more and more studies on the diagnosis and treatment bio-markers of depression ,7. Over Studies have shown that mice in a chronically stressed state display symptoms similar to those exhibited by patients with depression, such as anxious behavior, reduced socialization, and lack of pleasure (anhedonia) ,20. ThusStudies have demonstrated the role of miR-212 in neurological-related diseases . For exaWe hypothesized that miR-212 might play a role in depression via regulating NFIA. Hence, in the present study, we established a CUMS mouse model as previously described and performed an in-depth assessment of the miR-212-mediated mechanism in depression.C57BL/6 mice  were purchased from the Nanjing University Animal Research Center and received a standard diet of pellet chow. All experimental procedures were in accordance with the regulations of the Animal Use and Management Committee of the Wuhan Mental Health Center.3 from the left side of the cerebral cortex was preserved for flow cytometric detection of apoptosis, and the remaining tissue was placed into Eppendorf tubes® and frozen at −80°C for subsequent detection of relevant genes and proteins.In addition, animals had free access to water and were maintained on a 12–12 h light/dark cycle. The model was established over a 6-week period, during which mice were administered continuous stress and received different mild stresses each day to prevent anticipation and adaptation to the stress mode. The stressors included: (1) food deprivation for 24 h, (2) water deprivation for 24 h, (3) overnight lighting, (4) cage without wood chips for 24 h, (5) wetting wood chips with water for 24 h, (6) forced swimming in 8°C for 5 min, (7) tail clamping (1 cm from tail tip), (8) physical restraint for 6 h, and (9) 45° cage tilt with vertical axis for 3 h. After treatment, the mice were anesthetized with chloral hydrate and then decapitated. Brain tissues and peripheral samples were quickly harvested on ice and washed with phosphate-buffered saline (PBS), with brains dissected on ice dishes. Approximately 3 mmTo examine the role of miR-212 in depression, CUMS mice were divided into the following groups: control group (unstressed regular feeding), CUMS group (CUMS treatment), CUMS + mimic control treatment group , CUMS + miR-212 mimic treatment group , CUMS + miR-212 mimic + control-plasmid treatment group , CUMS + miR-212 mimic + NFIA-plasmid treatment group , and positive control group .Sucrose preference is an indicator of the animals’ preference for sugar water and the pursuit of ‘pleasure’, indicating that the animal was in a more rational state physically and mentally. A double bottle feeding method was used . To prevent mouse dependence on sucrose water, bottle positions were exchanged every 12 h. After 72 h, the consumption of sucrose and drinking water was measured, and sucrose preference was calculated as follows: Sucrose preference (%) = sucrose water consumption/(sucrose water consumption + pure water consumption) × 100% .The test animals were placed in a 24 cm high, 12 cm diameter Plexiglas cylinder tank, with a water depth of 20 cm and a water temperature of 25 ± 2°C. A suitably placed camera recorded the animal swimming for 6 min, and the cumulative immobility time was measured for the next 4 min. Animals were considered immobile when they stopped struggling and floated in the water for 2 s. Animals were acclimatized in the behavioral laboratory for at least 1 h before each experiment, ensuring that the time was consistent for each experiment. At the end of the experiment, body temperature was restored using a heat lamp, and the animals were then returned to their respective cages .The mice were suspended upside down by tape at a height of 30 cm, approximately 1 cm from the tail tip, to ensure that there was no possibility of climbing and grasping from front to back. Mice were considered immobile when completely motionless. Using a camera, the experiment was recorded for 6 min, and the cumulative immobility time was measured over the next 4 min. Animals were acclimatized in the behavioral laboratory for at least 1 h before each experiment, ensuring that time was consistent for each experimen .−ΔΔCt method [TRIzol reagent was used for total RNA extraction from peripheral blood samples and hippocampal tissues. Total RNA was measured using the OneDrop spectrophotometer . RNA was reverse transcribed to cDNA, and PCR amplification was performed using qPCR SYBE Green Master Mix on an ABI Step One Plus system. Briefly, amplification cycles were as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec, final extension at 60°C for 60 s. The primer sequences  were as follows: miR-212 forward, 5ʹ-GCCTCCTGACTCCAGGTCC-3ʹ, and reverse, 5ʹ-GCGCAAAGTGACTGGATGAA-3ʹ; U6 forward, 5ʹ-CTCGCTTCGGCAGCACATATACT-3ʹ, and reverse, 5ʹ-ACGCTTCACGAATTTGCGTGTC-3ʹ; NFIA forward, 5ʹ-ACCAGCTCAAAAAACCTGTGGA-3ʹ, and reverse, 5ʹ-TGTTGTGAAACGAAACACCCC-3ʹ; GAPDH forward, 5ʹ-CATCAAGAAGGTGGTGAAGCAG-3ʹ, and reverse, 5ʹ-CGTCAAAGGTGGAGGAGTGG-3ʹ. Target gene expression was quantified using the 2t method Briefly, 100 mg of hippocampal tissues were placed in fetal bovine serum (PBS) and homogenized thoroughly. Then, the supernatant was collected by centrifugation at 5000 × g at 4°C for 5 min. Cytokine levels  were measured using instructions provided by the corresponding ELISA kits.3 fresh cerebral cortex was digested with 0.25% trypsin without Ethylene Diamine Tetraacetic Acid (EDTA) for about 30 min to obtain a single-cell suspension. The cell density was adjusted to approximately 5 × 105, and the corresponding reagents were added according to the instructions provided by the Annexin V-FITC/PI Apoptosis Detection Kit. The reaction was performed at room temperature for 10 ~ 15 min, and the cells were detected on the flow cytometer within 1 h.In brief, 3 mmTotal proteins were extracted from neuronal cells using radioimmunoprecipitation assay (RIPA) lysis and measured using the bicinchoninic acid (BCA) method. In brief, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to separate proteins, then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skimmed milk for 50 min, membranes were incubated with primary antibodies  at 4°C overnight. After 24 h, the membranes were incubated with secondary antibody  and enhanced chemiluminescent reagent. Finally, Chemiluminescence Gel Imaging System and ImageJ software  were employed to analyze bands relative to the internal control.2 and at 37°C. The targeted fragment of NFIA in miR-212 was cloned from the genomic DNA by bioinformatics and then inserted into the luciferase reporter gene plasmid (psiCHECK-2) to construct the NFIA-wild type (wt) plasmid. In addition, the corresponding mutant control-plasmid NFIA-mut was prepared and purified by GenePharma . In 293 T cells, the reporter plasmids were co-transfected with miR-212 mimic or mimic control for 48 h. Finally, the luciferase activity was measured and evaluated relative to Renilla activity.Briefly, 293 T cells were purchased from American Type Culture Collection  and cultured in Dulbecco’s Modified Eagle Medium with 10% PBS and 1% penicillin-streptomycin under 5% COData values are expressed as mean ± standard error (SD) and analyzed using SPSS 22.0 . Student’s t-test or one-way ANOVA was applied for analyzing the data. A P value <0.05 was established as statistically significant.To confirm the underlying mechanism of miR-212 in depression, we first established CUMS-induced depression mice. As shown in Next, we examined the role of miR-212 in depression-like behaviors in CUMS mice. As shown in To explore the role of miR-212 in neuronal cell apoptosis in CUMS mice, after extraction of neuronal cells from the hippocampus of CUMS mice, the effects of miR-212 on neuronal cell apoptosis were examined using flow cytometry and Western blotting. As shown in To explore the role of miR-212 in CUMS-induced inflammatory response in the hippocampus, the inflammatory response was quantified by measuring hippocampal expression levels of inflammation-related cytokines in different groups. As shown in TargetScan software predicted the targeted sites between miR-212 and NFIA .FigureGain-of-function experiments further demonstrated the regulatory mechanism of the miR-212/NFIA axis in depression. After establishing the CUMS model, mice were intraperitoneally administered miR-212 mimic, control-plasmid, and NFIA-plasmid. As shown in Depression is characterized as a disorder of higher neurological functions in the brain. Based on accumulated evidence, organic changes in the central nervous system in patients with depression, and the hippocampus, as part of the limbic system, are responsible for mood regulation and cognitive management functions ,42. NumemiRNAs are a class of non-coding, single-stranded small molecule RNAs, approximately 22 nucleotides length, widely involved in regulating cell proliferation, differentiation, and apoptosis by regulating mRNAs . It has However, there were some limitations of current study. First, we did not analyze the expression of miR-212/NFIA in patients with depression, and the correlation between miR-212/NFIA expression and clinicopathological parameters in patients with depression. In addition, we did not study the effect of NFIA alone on mice with depression. We will perform these issues in the future.miR-212 might play a protective role in depression by regulating apoptosis and inflammatory responses in mouse hippocampal neurons by targeting NFIA. Thus, miR-212 may be a promising diagnostic and therapeutic target for depression clinical treatment.


Optic Neuritis is rare in Lyme borreliosis. The current knowledge of optic nerve involvement in Lyme borreliosis relies solely on case reports. The aim of this systematic review was to characterize and investigate the associated factors of optic neuritis in Lyme borreliosis. We further presented a very rare case of isolated bilateral optic neuritis in a Lyme seropositive patient. Borrelia burgdorferi, is the most frequent reported vector-borne disease in the United States [Lyme borreliosis, caused by d States , 2. Steed States . Lyme bod States . Neurolod States –7. Howevd States .. Ocular involvement is usually seen in the 2nd or 3rd stage of the disease. Although relatively uncommon, it could manifest multifariously such as conjunctivitis, keratitis, uveitis, periorbital oedema, cranial nerve II, III, IV, VII palsies, papilledema, reversible Horner’s syndrome, cotton wool spot, vascular occlusion, and optic neuritis [Kauffmann & Wormser 1990) was first to describe a case which the uniocular uveitis progressed to panedopthalmitis and loss of vision due to Lyme borreliosis [90 was fineuritis . Optic nThe goal of this systematic review was to characterize and investigate the associated factors of optic neuritis in Lyme borreliosis. We further presented a very rare case of isolated bilateral optic neuritis in a Lyme seropositive patient.A systemic review of Lyme optic neuritis cases characteristics was performed according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) statement. The electronic database Google Scholar was the primary source for article identification and PubMed was used for supplement. Articles were searched from database inception to July 2021 and identified through Keywords “Optic Neuritis”, paired with “Lyme disease”, “Lyme Borreliosis”, “Case report” and “Erythema Migrans”. MeSH term “Lyme Disease” paired with “Optic Neuritis” was used for search in PubMed. Accessible articles in English language were appraised and assessed via case report guidelines (CARE) by one individual and reference lists were scanned for additional studies of potential relevance.Articles that include elements of clinical assessment/diagnosis of optic neuritis and Lyme borreliosis (positive 2 tier serology tests), therapeutic interventions and outcome were included. Demographics, clinical findings, treatment, and treatment outcomes are listed in Table We also presented a very rare case of isolated bilateral optic neuritis in a Lyme seropositive patient. The written consent was obtained from the patient to present her illness.A 48 years old female with the past medical history of multiple sclerosis (MS), presented to her primary care physician in December 2019, with fever and sore throat. Three weeks later, patient returned and reported development of photophobia, eye pressure sensation, blurry vision, pain with eye movements for more than 3 weeks and noted central scotoma during the morning prior to her visit. Patient’s MS was first diagnosed in 2006. She has had relapses in 2010 and 2018, which both mainly presented as fatigue and difficulties in walking. Patient was on Copaxone (2006–2009), Gilenya (2010–2018) and currently treated with Ocrelizumab.She did not report any new neurological deficit except blurry vision. Her fundus exam and optical coherence tomography (OCT) revealed bilateral disc edema, and peripapillary retina nerve fiber layer thickening OU Fig. . Her visCSF analysis revealed mononuclear pleocytosis, and elevated glucose. CSF flow cytometry showed 27% neutrophils, 40% lymphocytes and 33% monocytes  was 20/20 OD, 20/25 OS, color vision, visual field and extraocular movement were all full. There was no signs or symptoms of optic neuritis.A total of 656 reports were retrieved by searching through Google Scholar and PubMed. After removing the duplicates and adding the manually tracked citations, 649 titles and abstracts were screened. Among the reviewed full texts, 33 reports were candidates of being included in this review and assessed for eligibility. We excluded 23 reports due to lack of confirmatory positive western blot following a positive ELISA test (15 reports), other etiology leading to optic neuritis (2 reports), no English version of the text (3 reports), and diagnosis of optic neuropathy other than optic neuritis (3 reports) , 5 were male and 6 were female. Seven cases were from Europe and 5 were from North America. The most common symptoms reported are related to optic neuritis – blurry vision (11 cases), headache (7 cases), scotoma (3 cases) and painful ocular movement (3 cases). Besides visual complaints, 4 reported neurological symptoms – paresthesia (3 cases) and ataxia (1 case); 3 reported arthralgia; and 3 reported nonspecific symptoms – fatigue, weakness, and myalgia.The most common signs found are bilateral optic disc edema (8 cases) and relative afferent pupillary defect (2 cases). Erythema migrans was diagnosed in 2 of the total patients. Eight patients did not recall having tick bites. Moderate vision loss (better than 20/200) was observed with majority of the patients (9 cases).Ten out of 11 patients have CSF study  has been shown to be effective in treating Lyme borreliosis . AntibioFinally, we present a case demonstrated a strong causal relationship of optic neuritis and Lyme borreliosis. Our patient’s optic neuritis could be reflective of her diagnosis of relapsing remitting multiple sclerosis. However, her presented symptoms were atypical for the patient’s MS due to absence of other neurological symptoms, and atypical compared to her flare ups in the past. Additionally, optic neuritis from multiple sclerosis is usually unilateral with normal or mild pupillary disc edema , 61, 62.Clinicians working in the endemic areas should consider Lyme borreliosis in patients presents with bilateral optic nerve head swelling, and painless progressive visual loss. Inadequate early treatment of Lyme borreliosis increases the likelihood of late manifestation and leads to relapses. Lyme borreliosis patients with optic neuritis respond well to antibiotics and have good prognosis.


Bone morphogenetic proteins (BMPs), which compose the largest group of the transforming growth factor-β (TGF-ß) superfamily, have been implied to play a crucial role in diverse physiological processes. The most intriguing feature of BMP signaling is that it elicits heterogeneous responses from cells with equivalent identity, thus permitting highly context-dependent signaling outcomes. In endothelial cells (ECs), which are increasingly perceived as a highly heterogeneous population of cells with respect to their morphology, function, as well as molecular characteristics, BMP signaling has shown to elicit diverse and often opposite effects, illustrating the innate complexity of signaling responses. In this review, we provide a concise yet comprehensive overview of how outcomes of BMP signaling are modulated in a context-dependent manner with an emphasis on the underlying molecular mechanisms and summarize how these regulations of the BMP signaling promote endothelial heterogeneity. Endothelial cells (ECs) exhibit great heterogeneity , in theiAs one of the archetypal receptor-ligand-mediated signaling pathways, bone morphogenetic protein (BMP) signaling has gained increasing importance in the development and homeostasis of the vascular system. Its ability to selectively activate ECs in a context-dependent manner without influencing the survival of ECs  satisfieTo better understand how BMP signaling modulates endothelial heterogeneity, it is essential to understand how BMP signaling functions within ECs. However, a simplified schematic diagram fails to capture the complex molecular landscape of BMP signaling, since distinct expression patterns of individual BMP signaling components result in diverse context-dependent outcomes . In addiAlk3 within naïve ECs led to the failure of venous specific marker expression in presumptive venous ECs  and lymphatic ECs (LECs), the two major subtypes of ECs . TherefoEven within BECs, it has been shown that the responsiveness toward BMP signaling also varies between arterial and venous ECs. For instance, we and others have shown that pro-angiogenic BMP signaling could selectively induce angiogenic responses in venous ECs without affecting arterial ECs . TogetheBone morphogenetic protein signaling seems to be highly context-dependent, eliciting opposite outcomes in similar cell types . It appein vivo, computational analyses revealed that cells distinguish distinct signaling environments, such as the concentration of ligands and differences in ligand–receptor complexes, to respond to BMP stimulation. Interestingly, inside the signaling-receiving cells, the canonical downstream target SMAD1/5/8 is activated regardless of the signaling-conferring ligand–receptor complex, suggesting the possibility that signaling-receiving cells are only capable of perceiving the sum of BMP ligand stimulation  of SMAD1 . In addiof SMAD1 . Moreoveof SMAD1 . Furtherof SMAD1 . Recent of SMAD1 . TherefoPost-translational modifications (PTMs) have been shown to contribute to the extensive versatility and complexity of signaling pathways in eukaryotic cells. Perturbations of PTMs have been linked to various diseases, illustrating the significance of PTMs in maintaining cellular homeostasis . While PIn addition to phosphorylation, ubiquitination and sumoylation, both of which are reversible PTM, have been shown to modulate the context of BMP signaling  Figure . The priO-linked N-acetyl-glucosamine (O-GlcNAc) moiety to target proteins, has been shown to fine-tune Drosophila BMP type I receptor Saxophone (Sax), an ortholog for human ALK2 , which contributes to the development of vascular lesions (Numerous studies have suggested that dysregulation of BMP signaling in ECs could lead to various diseases. For instance, mutations in BMPR2 predispose to the onset of PAH , a rare CCMs . DeletioCCMs . Moreove lesions , hinting lesions . In addi lesions . TherefoAs we obtain comprehensive knowledge on the role of BMP signaling in endothelial heterogeneity, we are confronted with a plethora of enigmas. Increasing evidence indicates that the complexity of BMP signaling dynamics provides essential regulation on endothelial plasticity. Emerging reports have identified novel fine-tuning regulatory mechanisms for BMP signaling. While their functions remain largely unknown in the endothelium, given the ubiquitous expression of these regulators, it is plausible that BMP signaling in ECs is similarly modulated. Since the role of BMP signaling in endothelial heterogeneity is still in its infancy, it will be of great importance to elucidate distinct regulatory mechanisms and more importantly its impact on the endothelial phenotype, which ultimately will lead to distinct physiological responses in the body. The plethora of regulatory possibilities provide an additional dimension for regulating distinct BMP signaling components and therefore further enrich the impact of the BMP signaling on endothelial heterogeneity. Not yet identified environmental factors, as well as various PTMs, might give us answers to yet unsolved enigmas and offer novel strategies to overcome limitations faced so far. Therefore, further investigations will provide insights into how such a conceptually simple signaling cascade can give rise to exquisite plasticity and versatility. Moreover, the availability of novel techniques, such as NGS, single-cell analysis, and bioinformatics, will refine our understanding of endothelial dysfunctions and concomitant diseases and ultimately contribute to the development of new therapeutic interventions for various diseases by fully harnessing the heterogeneity and context-dependent properties of BMP signaling.All authors wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Second, a randomization step is applied to the expanded function such that the leaked information is protected. We implement this expand-and-randomize coding scheme with two algebraic structures—the finite field and the modulo ring of integers, where the expansion step is realized with the addition operation and the randomization step is realized with the multiplication operation over the respective algebraic structures.We consider the secure computation problem in a minimal model, where Alice and Bob each holds an input and wish to securely compute a function of their inputs at Carol without revealing any additional information about the inputs. For this minimal secure computation problem, we propose a novel coding scheme built from two steps. First, the function to be computed is  Cryptographic primitives are canonical and representative problems that capture the key challenges in understanding the fundamentals of security and privacy, and are essential building blocks for more sophisticated systems and protocols. There is much recent interest in using information theoretic tools to tackle classical cryptographic primitives ,4,5,6,7.feasible, i.e., with at least three users, any function can be computed securely in the information theoretic sense [optimally, i.e., efficient secure computation solutions are not known for most cases [Secure computation refers to the problem where a number of users wish to securely compute a function on their inputs without revealing any unnecessary information. Interestingly, challenging as it seems, secure computation is always ic sense ,10. We cic sense ). Howevest cases .Z that is independent of the inputs and send codewords The main motivation of this work is to make progress towards constructing efficient secure computation codes. Towards this end, we focus on a minimal model of secure computation, introduced by Feige, Kilian, and Naor in 1994 . In thisstrong and optimality results remain weak, i.e., any function f can be computed securely while the construction of efficient codes remains open in general [The key feature of this formulation is that the communication protocol consists of only one codeword from each party that holds the input (thus non-interactive), while for the general secure computation formulation ,10, inte general ,13. In t general , but not general ,15,16, c general ,18,19,20 general , and unr general .The main contribution of this work is a novel coding scheme that relies on algebraic structures to ensure correctness and security. To illustrate the idea of our coding scheme, let us first consider an example. Suppose Alice and Bob each holds a ternary input, expand it to a linear function so that it becomes simpler to deal with. As shown in randomization so that the leaked information by the expanded function becomes confusable and thus protected. For this equality function example, when randomized expanded function does not reveal any additional information beyond the original equality function. The above expand-and-randomize procedure can be easily converted to a distributed secure computation protocol. In particular, Alice and Bob share a common random variable, z are independent, z is uniform over z) do not leak any information. Specifically, the joint distributions of As the equality function may not be easily computed in a secure manner, we first confusable sets over the finite field q is a prime power. The confusable function outputs turn out to be characterized by the property that their discrete logarithms  have the same remainder in modular arithmetic. Details will be presented in A closer inspection of the above scheme reveals that the key is to find an expanded function such that the expanded function outputs corresponding to the same original function output can be randomized to be fully confusable. The first main result of this work is to characterize the structural properties of such n, n arithmetic. Let us consider an example to illustrate how f is OFF and the output is 0 . When f is ON and the output is equal to the input vector , they will produce identically distributed sets of values; specifically, both will produce the confusable set From this example, we find that the crux of the scheme is a e set S.Z6={0}∪{1p is used. Note that for a prime p, the algebraic operations (addition and multiplication) in both finite field p, Our proposed coding scheme is inspired by two examples  presented in Appendix A and Appendix B of the original minimal secure computation paper , where m from in  to a genf is discrete, and use f. Z whose distribution does not depend on Consider a pair of inputs Z, and has Z, and has f is known to Alice, Bob, and Carol. As our proposed code will have a fixed length, here we only define fixed-length codes, i.e., Alice and Bob wish to compute From A rate tuple L-length extensions (block inputs) will be considered when they play more significant roles in the results, e.g., when we discuss The main result of this work is a new achievable scheme for secure computation and the new scheme works for any joint distribution of n, denoted as In this section, we present a novel secure computation code that implements the expand-and-randomize scheme over the finite field Definition 1 (Confusable Sets and Randomizer).Sets The requirement in Definition 1 is stronger than what is needed for security. It suffices to have identical (instead of uniform) distributions over some disjoint set (instead of the confusable set). However, for our proposed scheme, it turns out that these relaxations do not lead to improved achievable rate regions such that they are not considered for simplicity. The notions introduced in Definition 1 are closely related to group actions (and orbits) studied in group theory . Along tDefinition 2 (Feasible Expanded Function).For a function f is the equality function and f is mapped to f is mapped to For an example of a feasible expanded function  over A feasible expanded function as defined above naturally leads to a correct and secure computation scheme, presented in the following theorem.Theorem 1.For any function where Proof of  Theorem 1.z. Therefore, The proof of correctness and security follows in a straightforward manner from the definitions of the confusable sets, the randomizer, and the feasible expanded function. First, we consider the correctness constraint. To recover nstraint . Considen case is considered in The coding scheme in Theorem 1 relies on the structure of the confusable sets and the randomizer upon which feasible expanded functions are built. Thus, it is crucial to understand the structure of the confusable sets and the randomizer, i.e., which set of elements can be used as the randomizer and how the algebraic object is partitioned to confusable sets. This structure problem is addressed next, through algebraic characterizations. The finite field case is considered in p is a prime and n is a positive integer. the finite field. The n that always exists. The non-zero elements of We first recall some basic facts of finite fields (refer to standard textbooks such as that in ). A finiExample 1.The finite field Equipped with the above results d+i−1}={gi−1,gdTo facilitate the presentation of the algebraic characterization of the confusable sets and the randomizer over Definition 3 (Set of Integers with Same gcd).Consider any proper divisor d of a given integer n, i.e., n (it turns out to form a group under multiplication modulo n), so we adopt the standard existing notation For example, suppose ee e.g., ) and numee e.g., ), and isNote thatH.We present an important result on the projection of a multiplicative subgroup of Lemma 1.Consider an arbitrary subgroup G denotes a subgroup and the subscript specifies the original group. The proof of Lemma 1 is presented in In Lemma 1, Example 3.Consider a subgroup Consider another subgroup Consider d as Given a subgroup ter 3 of  or Theorter 3 of ). Settining with , i.e., ZExample 4.Continuing from Example 3, consider a subgroup where the partition is obtained from the cosets, e.g., For another choice of For the final choice of d is a feasible choice of the confusable sets. This result is stated in the following theorem.The collection of the cosets Theorem 3.For Before presenting the proof of Theorem 3, we first give an example to illustrate its meaning.Example 5.Continuing from Example 4, consider a subgroup For each of the confusable set above, it is easy to verify that when an element is multiplied with γ . Interestingly, when the size of the underlying field or ring is the same as the input size, the scheme in Theorem 1 achieves the information theoretically optimal rate region. Without loss of generality, for secure computation problems, we assume there are no identical rows or columns in the function table .Proposition 1.Consider independent and uniform inputs, i.e., Achievability directly follows from Theorem 1 and converse obayashi  . One could start from the confusable sets of To the best of our knowledge, the only existing information theoretic converse results for the secure computation problem are the ones obtained in , whose eThe proof of Lemma 1 consists of two parts.d. Note that for finite groups, the verification of subgroups only requires the check of the closure property .g of d is a divisor of n, we have For (1), note that any element k. Then,For (2), consider any two elements of Second, we show that in the multiset d is commutative. Then, there exists From the first part of the proof, we know that Applying  to Gd an we have∀j∈{1,⋯,QComparing  and 52)52) , which Example 6.Consider the function From Theorem 1, Alice will send Interestingly, if we choose z to be uniform over Both Remark 3.If variable-length codes are allowed, then the above code can be further improved. Specifically, Alice does not need to distinguish whether rem 9 in  for any ented in  .For a general given function (such as ) and solHitherto, we have focused exclusively on scalar codes and zero-error schemes that work for any joint distribution of n coding ) that exExample 7.Consider the binary AND function Then, from Theorem 1, we set dix B of .We note that for correct decoding, Carol will compute n coding .The improvement of the communication rate comes from the observation that in our proposed code, we consider the worst case, i.e., U is not compressed and a symbol from  3/8 see  and the Next, we outline how to use structured linear source codes to achieve the rate tuple where the ‘+’ and ‘×’ operators are symbol-wise, and the ‘·’ operator is the matrix multiplication operator. Further optimizations of the common randomness consumption are possible, i.e., the same randomizer can be used for each input bit and it suffices to use an additive common randomness variable with entropy  channel , was use channel ), was us channel , and gen channel  and Rema channel ).The security constraint is easily verified. The scalar code is secure by Theorem 1. Then independent application of the scalar code to L-length extensions is also secure. Multiplying with a deterministic matrix A will not leak any information.Therefore, the optimized block code has vanishing probability of error and is secure. The achieved rate tuple is Finally, we note that the idea of using Korner–Marton coding for secure computation is not new, e.g., it has been applied to secure sum computations ,39. WhilRemark 4.Interestingly, the zero-error code presented above for AND computation is information theoretically optimal in terms of the communication rate (refer to Theorem 11 of ). That iior work ,39). We n is not a prime. While the same linear compression technique can be applied, the rate performance is not known. That is, it is not known how large the matrix A needs to be, if we wish to recover The above linear compression technique applies to all secure computation codes over e.g., in ,41 and rm and wish to securely compute if m is a prime power m is not a prime power? We may increase m to a prime power and then use the previous approach. This approach will require that Alice and Bob each sends a symbol of size larger than m (no matter whether m is a prime power or not). To this end, we need to use a variant of the expand-and-randomize scheme from Theorem 1. To illustrate the idea, in the following we consider the simplest example where m is not a prime power, i.e., In this subsection, we continue the discussion on the equality function in the introduction. We consider the equation function with arbitrary input size, i.e., Example 8.Consider Alice and Bob share a common random variable The ‘+’ and ‘×’ operations in computing We have zero error because Therefore, security is guaranteed and sending Remark 5.The above scheme generalizes in a straightforward manner to any integer m using a prime-power decomposition of m . As the achieved communication rate n. We characterized the algebraic structures of the feasible expanded functions through the notion of confusable sets. We find it interesting that while we consider only information theoretic security, the tools invoked from algebra and number theory arise frequently and lie in the core of cryptography under computational security . TheExample 9.Consider the function shown in Therefore, according to Theorem 1, it suffices to send a symbol from However, an improved rate tuple where Example 10.Consider the function shown in Therefore, according to Theorem 1, it suffices to send a symbol from However, an improved rate tuple is achievable using a different coding scheme. To see this, we assume that thm 3 of , which iwhere n, it is interesting to explore other widely studied algebraic objects in abstract algebra [embedding the function to compute in another function that guarantees security. The potential of this general embedding theme remains to be fully explored. Finally, we note that while we focus on the basic model of minimal (non-interactive three-user) secure computation, the proposed scheme generalizes immediately to interactive protocols (by first interactively generating the common randomness) and to more users . Exploration of the proposed scheme to various models of secure computation [Going forward, while the characterization of the algebraic structures of the confusable sets over  algebra , e.g., tputation  is an in


Serum uric acid (UA) has long been identified as a prognostic factor of adverse outcomes in pulmonary hypertension. However, there remains a paucity of evidence on patients with idiopathic pulmonary artery hypertension (IPAH) in the era of targeted drug therapy. This study aims to explore the impact of serum UA levels on the disease severity and mortality in patients with IPAH.Consecutive patients diagnosed with IPAH were enrolled, from which UA levels at baseline and the first follow-up were collected. Patients were divided into groups of “hyperuricemia,” which is defined as serum UA level ≥357 μmol/L in women and ≥420 μmol/L in men, and otherwise “normouricemia.” The potential relationship between UA and hemodynamics at right heart catheterization was investigated. Associations between UA and survival were evaluated by Kaplan-Meier analysis and Cox proportional hazard modeling.r = 0.47, p < 0.001) and higher pulmonary vascular resistance . During a median follow-up of 34 months, there were 32 deaths recorded, accounting for a 15.5% mortality rate. Patients with hyperuricemia had a significantly lower survival rate than those with normouricemia . Hyperuricemia at baseline was independently associated with a 2.6-fold increased risk of 5-year death, which was consistent across different subgroups, especially in females and those aged ≥30 years (each p < 0.05). Individuals with higher variability in UA had a higher mortality than those with stable UA .Of 207 patients with IPAH, 121 (58.5%) had hyperuricemia. Higher serum UA levels were associated with lower cardiac index (Baseline hyperuricemia and high variability in serum UA at first follow-up were related to a higher rate of 5-year mortality in patients with IPAH. Closely detecting the UA levels may aid in the early recognition of IPAH patients at higher mortality risk. Pulmonary artery hypertension (PAH) is a devastating condition characterized by progressive remodeling in the pulmonary vasculature, leading to increased pulmonary resistance, right ventricular failure, and even death . DespiteSerum uric acid (UA), the end product of purine degradation, has been proposed as a prognostic biomarker in PAH , 6 and oTherefore, this study aimed to investigate the associations of serum UA levels and hemodynamic parameters, and analyze the prognostic impact of baseline hyperuricemia and the variability of serum UA on long-term mortality in patients with IPAH.Of the 211 consecutive patients diagnosed with IPAH in our institute between January 2015 and January 2020, three patients were excluded due to the absence of serum UA data, and one was excluded due to lung transplantation. The remaining 207 patients with IPAH were enrolled in the current study. These patients' demographic data [including body mass index (BMI)], World Health Organization (WHO) functional classification, primary laboratory results, serum UA levels, and hemodynamic parameters derived from RHC were gathered and analyzed. The study was performed with the approval of the Fuwai Hospital Ethics Committee (No. 2018-1100). Written informed consent was obtained from each patient included in this study.Patients underwent diagnostic RHC while in stable condition during hospitalization. A baseline hemodynamic profile comprised of mean pulmonary arterial pressure (mPAP), right atrial pressure (RAP), and pulmonary vascular resistance (PVR) were measured at end-expiration. The cardiac output was calculated using the Fick method, and the cardiac index was obtained by dividing the cardiac output by the body surface area. Following 2015 ESC/ERS guidelines , all stuBlood samples for baseline measurements, including serum UA were obtained from a peripheral vein after an overnight fast upon admission. After that, medications including diuretics, cardiac inotropes, and targeted drugs were initiated based on an adequate risk status assessment. This was to reduce their influence on baseline serum UA levels to a large extent. Serum UA levels were determined using the uricase-peroxidase method. Considering the sex difference in serum UA levels, hyperuricemia was defined as baseline UA levels >357 μmol/L in women and 420 μmol/L in men, consistent with previous studies . The othTo explore the potential impact of variation in UA levels on the prognosis, patients with a second measure of serum UA at the first follow-up within 3 months were retrospectively investigated. Similarly, patients were classified into hyperuricemia and normouricemia groups according to their UA levels at first follow-up. An increase or a decline in UA levels was thereby noted. The serum UA variability (UAV) was the exposure variable of interest. It was defined as the fluctuating amplitude of serum UA levels as compared with the baseline UA level. The median of UAV was 53 μmol/L and was applied to classify patients into a high UAV group  and a low UAV group .The primary outcome of the current study was the rate of 5-year all-cause mortality. Telephone follow-up was used to monitor clinical outcomes every 3–6 months. Patient deaths were identified by medical records or death certificates review. Survival of IPAH patients was estimated from the date of the first catheterization until 5 years or to the date of patient death if that occurred first. One patient who underwent transplantation was removed from the analysis at the time point.t-tests for continuous variables and Pearson's x2 tests for categorical variables. A non-parametric Mann-Whitney U-test was used when data were not normally distributed. Univariate correlation analysis and multiple stepwise linear regression analysis were used to investigate potentially related factors of serum UA levels. Survival curves were derived using the Kaplan-Meier method and were compared using the log-rank test. The independent association of hyperuricemia with mortality was tested by multivariate Cox proportional hazards regression analysis. A p-value of <0.05 was required for statistical significance in all cases. Statistical analyses were conducted using R statistical version 3.6.3  within RStudio statistical software version 1.1.453.Continuous variables are presented as the mean ± standard deviations or as the medians (interquartile range) after evaluation of normality. Categorical variables are presented as counts and percentages. Comparisons of variables between the hyperuricemia and normouricemia groups were made by two-tailed independent sample 2) were included in this study. The majority was classified as WHO functional class II and III, consisting of 103 and 90 individuals, respectively. The detection levels of serum UA ranged from 121.5 to 929.7 μmol/L. Hyperuricemia was present in 121 patients (58.5%) with IPAH. Approximately 85.5% of participants received targeted therapy for PAH at the index hospitalization. The remaining refused to take mainly due to financial burden, cautions about adverse effects, and intolerability.A total of 207 patients with IPAH  and normouricemia group  were summarized in r = 0.36, p < 0.001), mPAP , and RAP , and negatively correlated with cardiac index . Serum UA levels did not significantly correlate with age (p = 0.436) but were significantly associated with the cardiac index, NT-proBNP, creatinine, and BMI in univariate Pearson's correlation analysis (p = 0.005), BMI , creatinine , age , and NT-proBNP   .p = 0.028). There were 32 deaths in the entire cohort over the study period, accounting for a 15.5% mortality rate. Twenty-six deaths occurred within the hyperuricemia group and six within normouricemia group (p = 0.003).Comparison of baseline serum UA levels in survivors and non-survivors demonstrated a significantly higher median UA than in non-survivors   .p = 0.019), patients aged ≥30 years , and patients with RAP ≥4 mmHg .In stratified analyses according to gender, age, WHO functional classes, and severity of IPAH based on the median value of hemodynamic parameters, the impact of hyperuricemia on the hazard of mortality was plotted . The incp = 0.91). A changing tendency, i.e., an increase or decrease in UA levels, also yielded negative results (p = 0.23). Interestingly, IPAH patients with a high UAV, compared with those with low UAV, are associated with an increased all-cause mortality rate (p = 0.023).The second measure of serum UA (412.6 ± 126.7 μmol/L) at first follow-up was available in a subset of 150 patients. As shown in the Kaplan-Meier curves in Serum Uric Acid and Long-Term Mortality in IPAH Patients , there wIn the present study, we investigated the utility of serum UA as a predictor of disease severity and long-term mortality in patients with IPAH. The main findings were that (1) a relatively high prevalence of hyperuricemia was observed in patients with IPAH (58.5%); (2) serum UA levels at baseline positively correlated with mPAP, RAP, and PVR, while negatively correlated with a cardiac index which remained significant after adjusting for potential confounders; (3) patients with hyperuricemia at baseline and those with high variability in serum UA at first follow-up had a significantly lower survival rate than those with normouricemia during follow-up of 5 years; and (4) hyperuricemia was independently associated with the 5-year mortality, and the association consisted in specific subgroups.In the last guidelines for PAH risk stratification at baseline and during follow-up, the only two biomarkers that function as dynamic measures are the NT-proBNP and the brain natriuretic peptide (BNP) . NeverthAmong a range of hemodynamic parameters, the cardiac index remained the most vital related factor of serum UA levels, and our study revealed an inverse relationship between serum UA levels and cardiac index. Cardiac index (the body-surface-area-adjusted cardiac output) was recorded and analyzed to avoid potential influence by variant body weight and height. The fundamental basis accounting for this phenomenon is attributed mainly to lower perfusion to the kidneys, which compromises the renal excretion of UA. Meanwhile, reduced perfusion to the lungs with a low cardiac index may also result in hypoxia, and therefore, accumulation of serum UA . Additioin vitro, UA can also stimulate platelet-derived growth factors and vasoconstrictors such as endothelin-1 and angiotensin II  and therapy decisions should be made individually in patients with IPAH. Presumably, combination therapy that targets multiple signaling pathways with or without specific UA-lowering agents may be appropriate to reduce disease progression and mortality risk in IPAH patients with a high baseline UA or variability. This speculation would be more robust if serum UA levels significantly improved the risk discrimination power of the simplified risk table . Howeverp = 0.825) and UA variability (p = 0.435) between treatment groups and whether with or without treatment was also not statistically significant. Regarding the effects of UA-lowering agents that were not available and collected in our study, a meta-analysis of 35 randomized controlled trials in patients with gout showed that UA-lowering therapy did not reduce the major adverse cardiovascular events, including the all-cause mortality . The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LY and ZH contributed to the study design, data analysis, and manuscript drafting and revision. ZZ and QZ critically reviewed and revised the manuscript. YT, YZ, XL, and AD performed literature search. QL and ZL provided professional advice on data interpretation, critically reviewed, and revised the manuscript. All authors contributed substantially to the work and agreed to submit the manuscript for publication.This study was funded by National Natural Science Foundation of China , Beijing Municipal Science and Technology Project (Z181100001718200), National Precision Medical Research Program of China (2016YFC0905602), Double First-Class Discipline Construction Fund of Peking Union Medical College and Chinese Academy of Medical Sciences (2019E-XK04-02), CAMS Innovation Fund for Medical Sciences (CIFMS) , Capital's Funds for Health Improvement and Research (2020-2-4033), and Beijing Municipal Natural Science Foundation (7202168).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.


Stigma Maydis) has been utilized as an important herb against obesity by Chinese, Korean, and Native Americans, but its phytochemicals and mechanisms(s) against obesity have not been deciphered completely. This study aimed to identify promising bioactive constituents and mechanism of action(s) of corn silk (CS) against obesity via network pharmacology. The compounds from CS were identified using Gas Chromatography Mass Spectrometry (GC-MS) and were confirmed ultimately by Lipinski’s rule via SwissADME. The relationships of the compound-targets or obesity-related targets were confirmed by public bioinformatics. The signaling pathways related to obesity, protein-protein interaction (PPI), and signaling pathways-targets-bioactives (STB) were constructed, visualized, and analyzed by RPackage. Lastly, Molecular Docking Test (MDT) was performed to validate affinity between ligand(s) and protein(s) on key signaling pathway(s). We identified a total of 36 compounds from CS via GC-MS, all accepted by Lipinski’s rule. The number of 36 compounds linked to 154 targets, 85 among 154 targets related directly to obesity-targets (3028 targets). Of the final 85 targets, we showed that the PPI network , 12 signaling pathways on a bubble chart, and STB network  are considered as therapeutic components. The MDT confirmed that two key activators  bound most stably to PPARA, PPARD, PPARG, FABP3, FABP4, and NR1H3 on the PPAR signaling pathway, also, three key inhibitors  bound most tightly to AKT1, IL6, FGF2, and PHLPP1 on the PI3K-Akt signaling pathway. Overall, we provided promising key signaling pathways, targets, and bioactives of CS against obesity, suggesting crucial pharmacological evidence for further clinical testing.Corn silk ( Obesity is a serious health issue worldwide because it is involved in the main causes of comorbidity and mortality, including diabetes, hypertension, heart failure, atherosclerosis, and some cancers ,2. ObesiIrcinia variabilis) [Dysidea villosa) [Stylissa flabelliformis) [Phorbas sp.) [Curcuma longa rhizome), Carnosic acid and carnosol , Epigallocatechin 3-O gallate (from Camellia sinensis), Ursolic acid (from Actinidia arguta root), and Crocetin and crocin (from Gardenia jasminoides fruits) [The most optimal therapeutic strategy against obesity is to inhibit the accumulation of fat in the body as well as to suppress the appetite with special medication ,9. At priabilis) , Dysidinvillosa) , Questiniformis) , and Phobas sp.) . Other r fruits) . Current fruits) . However fruits) . From th fruits) . Network fruits) . With th fruits) . Moreove fruits) . The aimCorn silk (CS) were collected from , Gyeongsangbuk-do, Korea, in July 2021. The CS were dried in a shady zone at room temperature (20–22 °C) for 7 days, and dried CS powder was made using an electric blender. Approximately 20 g of CS powder was soaked in 1000 mL of 100% ethyl alcohol  for 15 days and repeated 3 times to achieve a high yield rate. The solvent extract was collected, filtered with Whatman filter paper No. 1  and evaporated using a vacuum evaporator  at 40 °C. The yield after evaporating was 1.98 g (Yield rate: 0.99%), which was calculated as follows:m/z). The fragmentation patterns of mass spectra were compared with those stored in the W8N05ST Library MS database . The percentage of each compound was calculated from the relative peak area of each compound in the chromatogram [Agilent 7890A  was used to perform GC-MS analysis. GC was equipped with a DB-5 (30 m × 0.25 mm × 0.25 µm) capillary column . Initially, the instrument was maintained at a temperature of 100 °C for 2.1 min. The temperature rose to 300 °C at a rate of 25 °C/min and was maintained for 20 min. Injection port temperature and helium flow rate were ensured as 250 °C and 1.5 mL/min, respectively. The ionization voltage was 70 eV. The samples were injected in split mode at 10:1. The MS scan range was set at 35–900  to identify SMILES (Simplified Molecular Input Line Entry System) format. The screening of DLCs is based on Lipinski’s rule via SwissADME (http://www.swissadme.ch/) (accessed on 9 September 2021). Additionally, topological polar surface area (TPSA) to measure cell permeability of compounds was identified by SwissADME . Commonly, its cut-off value to evaluate cell permeability is typically less than 140 Å2 [The chemical constituents in CS were detected via GC-MS analysis, which were input into PubChem  and OMIM (https://www.ncbi.nlm.nih.gov/omim) (accessed 13 September 2021). The overlapping target proteins between DLCs from CS and obesity-related target proteins were identified and visualized on InteractiVenn [The bioactives confirmed by Lipinski’s rule put the SMILE format into two two public cheminformatics: Similarity Ensemble Approach (SEA) (accessed on 10 September 2021)  and Swisperiment ,32. AltoactiVenn . Then, whttps://string-db.org/, accessed 14 September 2021) [The interaction of the final overlapping target proteins was identified by STRING analysis (er 2021) . The numThe STB networks were visualized as a size map, based on a degree of value. In the network map, green rectangles (nodes) represented the signaling pathways; yellow triangles (nodes) represented the target proteins; red circles (nodes) represented the bioactives. The size of the yellow triangles stood for the number of relationships with signaling pathways; the size of red circles stood for the number of relationships with target proteins. The assembled network was constructed by utilizing RPackage.https://www.rcsb.org/, accessed 16 September 2021). The proteins were chosen as. PDB format were converted into. pdbqt through Autodock .The bioactives related to the two key signaling pathways were converted. sdf from PubChem into. pdb format utilizing Pymol, and thus they were converted into. pdbqt format via Autodock. The number of the six proteins on the PPAR signaling pathway, i.e., PPARA (PDB ID: 3SP6), PPARD (PDB ID: 5U3Q), PPARG (PDB ID: 3E00), FABP3 (PDB ID: 5HZ9), FABP4 (PDB ID: 3P6D), and NR1H3 (PDB ID: 2ACL), and the number of the seven proteins on PI3K-Akt signaling pathway, i.e., AKT1 (PDB ID: 3O96), IL6 (PDB ID: 4NI9), VEGFA (PDB ID: 3V2A), PRKCA (PDB ID: 3IW4), FGF1 (PDB ID: 3OJ2), FGF2 (PDB ID: 1IIL), and PHLPP1 (not available in the PDB) were identified on STRING via RCSB PDB  [The center of each target protein on PI3K-Akt signaling pathway was Akt1 , IL6 , VEGFA , PRKCA , FGF1 , FGF2 , PHLPP1 . The active site’s grid box size was x = 40 Å, y = 40 Å, z = 40 Å. The detailed information of 2D binding was identified by LigPlot2)  .A total of 429 target proteins from SEA and 466 target proteins from STP linked to the abovementioned 36 compounds were identified through SMILES format . The resAs shown in Using STRING analysis, 79 out of 85 target proteins were correlated closely with each other with 79 nodes and 357 edges . The eliThe results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that 85 target proteins were related directly to 12 signaling pathways . The 12 signaling pathways were implicated with occurrence and development of obesity, suggesting that these pathways might be important signaling pathways of CS against obesity. The description of the 12 signaling pathways was represented in A signaling pathway-target protein- bioactive (STB) network of CS was exhibited in Through MDT analysis, it was unveiled that PPARA(PDB ID: 3SP6) was associated with 9 bioactives: (1)β-Amyrone, (2) Squalene, (3) Ethyl palmitate, (4) Heneicosanoic, 2,4-dimethyl-,methyl ester, (5) Oleic acid, (6) Ethyl linoleate, (7) Palmitic acid, (8) Linoleic acid, and (9) (Z)-9-Hexadecenal, PPARD (PDB ID: 5U3Q) was related to 8 bioactives: (1) β-Stigmasterol, (2) β-Sitosterol, (3) Heneicosanoic, 2,4-dimethyl-,methyl ester, (4) Ethyl linoleate, (5) Linoleic acid, (6) Oleic acid, (7) Palmitic acid, and (8) (Z)-9-Hexadecenal, PPARG (PDB ID: 3E00) was connected to 6 bioactives: (1) β-Amyrone, (2) Ethyl linoleate, (3) Linoleic acid, (4) Oleic acid, (5) Palmitic acid, and (6) (Z)-9-Hexadecenal, FABP3 (PDB ID: 5HZ9) was associated with 12 bioactives: (1) β-Amyrone, (2) Heneicosanoic, 2,4-dimethyl-,methyl ester, (3) Eicosane, (4) Ethyl stearate, (5) Ethyl heptacecanoate, (6) Ethyl linoleate, (7) Ethyl palmitate, (8) Linoleic acid, (9) (Z)-9-Hexadecenal, (10) Oleic acid, (11) Palmitic acid, and (12) 5-Aminovaleric acid, FABP4 (PDB ID: 3P6D) was related to 11 bioactives: (1) β-Amyrone, (2) Heneicosanoic, 2,4-dimethyl-,methyl ester, (3) Ethyl stearate, (4) Ethyl palmitate, (5) Ethyl heptacecanoate, (6) Ethyl linoleate, (7) 5-Aminovaleric acid, (8) Oleic acid, (9) Linoleic acid, (10) Palmitic acid, and (11) (Z)-9-Hexadecenal, and NR1H3 (PDB ID: 2ACL) was connected to 9 bioactives: (1) β-Amyrone, (2) β-Stigmasterol, (3) β-Sitosterol, (4) Estradiol, 3-deoxy, (5) Sitostenone, (6) 24-epicampesterol, (7) Ethyl linoleate, (8) Linoleic acid, and (9) Oleic acid.It was observed that β-Amyrone had the highest affinity on five out of six target proteins: −16.1 kcal/mol on PPARA (PDB ID: 3SP6), −14.0 kcal/mol on PPARG (PDB ID: 3E00), −21.5 kcal/mol on FABP3 (PDB ID: 5HZ9), −13.2 kcal/mol on FABP4 (PDB ID: 3P6D), and −15.4 kcal/mol on NR1H3 (PDB ID: 2ACL). Interestingly, the highest affinity on PPARD (PDB ID: 5U3Q) was β-Stigmasterol with −10.8 kcal/mol. The docking detail information is enlisted in Through MDT analysis, it was revealed that AKT1 (PDB ID: 3O96) was related to a sole bioactive: (1) Neotocopherol, IL6 (PDB ID: 4NI9) was associated with 3 bioactives: (1) Xanthosine, (2) 2-Ethylacridine, and (3) Linoleic acid, VEGFA (PDB ID: 3V2A) was connected to (1) Ethyl palmitate, (2) Ethyl heptadecanoate, (3) Ethyl stearate, (4) Ethyl linoleate, and (5) α-D-2-deoxyribose, PRKCA (PDB ID: 3IW4) was linked to 9 bioactives: (1) Ethyl palmitate, (2) Ethyl heptadecanoate, (3) Ethyl linoleate, (4) 2,4,4-Trimethylpentane-1,3-diyl bis(2-methylpropanoate), (5) Linoleic acid, (6) Ethyl stearate, (7) (Z)-9-Hexadecenal, (8) Palmitic acid, and (9) Oleic acid, FGF1 (PDB ID: 3OJ2) was related to 4 bioactives: (1) Sitostenone, (2) 24-epicampesterol, (3) Cytidine, and (4) α-D-2-deoxyribose, FGF2 (PDB ID: 1IIL) was associated with 7 bioactives: (1) β-Amyrone, (2) β-Stigmasterol, (3) Sitostenone, (4) 24-epicampesterol, (5) β-Sitosterol, (6) Cytidine, and (7) α-D-2-deoxyribose, and PHLPP1 was linked to a sole bioactive: (1) Neotocopherol. It was observed that Neotocopherol on AKT1 (PDB ID: 3O96), Xanthosine on IL6 (PDB ID: 4NI9), and β-Amyrone on FGF2 (PDB ID: 1IIL) had the highest affinity among bioactives from CS as well as better affinity than positive controls. The docking detail information is enlisted in β-Amyrone, out of 36 bioactives from CS, was associated with the number of 6 target proteins on both the PPAR signaling pathway and the PI3K-Akt1 signaling pathway, considered as key signaling pathways of CS on obesity. Noticeably, it was unveiled that β-Amyrone (a triterpenoid derivative) on PPARA (PDB ID: 3SP6), PPARG (PDB ID: 3E00) and FGF2 (PDB ID: 1IIL) had better affinity than the positive controls. Likewise, the β-Stigmasterol on PPARD (PDB ID: 5U3Q) had better affinity than Cardarine, which is used as an anti-obesity drug. A report demonstrated that α,β-amyrin, as a triterpenoid derivative homologous to β-Amyrone, inhibits adipocyte differentiation by inactivating PPARG . AnotherBesides, our study provided that 11 out 12 signaling pathways associated with AKT1 might have inhibitory effects for the alleviation of obesity, including PI3K-Akt signaling pathway. In contrast, PPAR signaling pathway of CS on obesity is a sole activator mechanism, not related to AKT1. According to a bubble chart, PPI, and STB networks results, we identified 2 signaling pathways, 13 targets, and 27 bioactives, and thus MDT verified that 4 bioactives  among 27 bioactives could stably bind to the targets, indicating that CS might activate the PPAR signaling pathway, and inactivate PI3K-Akt signaling pathway. Moreover, the final 4 bioactives have better stable affinity than the positive controls. To sum things up, we adopted 2 key signaling pathways , 10 targets , and 4 bioactives  see . We remoOverall, this study demonstrated the potential signaling pathways, targets, and bioactives in treating obesity based on network pharmacology analysis. We identified 2 key signaling pathways , 13 targets , and 4 bioactives  of CS against obesity. A total of 10 out of 13 targets have better affinity or valid value in comparison with the positive controls: PPARA, PPARD, PPARG, FABP3, FABP4, NR1H3, AKT1, IL6, FGF2, and PHLPP1. The AKT1 with the highest degree value was considered as the uppermost target, Neotocopherol was a critical bioactive that was bound most stably to AKT1. Notably, β-Amyrone as an activator could dock well with PPARA, PPARG, FABP3, FABP4, NR1H3 on the PPAR signaling pathway, in contrast, β-Amyrone as an inhibitor could dock stably with FGF2 on the PI3K-Akt signaling pathway. This study shows that β-Amyrone of CS might have dual-efficacy to alleviate obesity. To conclude, we described the therapeutic evidence to expound key signaling pathways, targets, and bioactives of CS against obesity. However, there are still limitations to our analysis, which needs to be further improved, through either in vitro or in vivo. Last but not least, our analysis did not consider the expression of the target gene practically after treating the selected compounds, which should be implemented in the future.


Wnt signaling pathways constitute a group of signal transduction pathways that direct many physiological processes, such as development, growth, and differentiation. Dysregulation of these pathways is thus associated with many pathological processes, including neurodegenerative diseases, metabolic disorders, and cancer. At the same time, alterations are observed in plasma membrane compositions, lipid organizations, and ordered membrane domains in brain and metabolic diseases that are associated with Wnt signaling pathway activation. Here, we discuss the relationships between plasma membrane components—specifically ligands, (co) receptors, and extracellular or membrane-associated modulators—to activate Wnt pathways in several brain and metabolic diseases. Thus, the Wnt–receptor complex can be targeted based on the composition and organization of the plasma membrane, in order to develop effective targeted therapy drugs. Wnt signaling pathways are highly conserved in the animal kingdom, based on their components and functional roles in the regulation of development, tissue homeostasis, and regeneration ,5,6,7,8.The plasma membrane plays a fundamental role in the regulation of cell signaling. Regulation occurs through the surface receptors, modulators, and associated lipids that actively control the transmission of molecular signals from the outside to the inside and activate downstream signaling events. The plasma membrane consists of nanodomains—the so-called ordered membrane domains or lipid rafts that are defined as dynamic assemblies of various saturated lipids, sterols, glycosphingolipids, and glycosyl-phosphatidylinositol (GPI)-anchored proteins ,13,14. T2+ pathways , calcium/calmodulin-dependent protein kinase type II (CaMKII), and nuclear factor of activated T cells (NFAT), and regulates cell movement, cell fate, and cell migration as well as suppressing the canonical Wnt pathway renin receptor ATP6AP2 in caveolae/lipid raft microdomains is essential for neuronal differentiation of stem cells, with a concomitant transition from Wnt/β-catenin to Wnt/PCP signaling, and that these domains may be used as a potential target for the treatment of neurodegenerative disorders . In a grntiation . The glyntiation . Palmitontiation ,56,57. Sntiation ,58. UndeAlzheimer’s disease (AD) is a neurodegenerative disorder that accounts for two-thirds of dementia cases ,60,61; iThe secreted Wnt antagonist Dkk1 was found to be highly expressed in the brains of Alzheimer’s patients and in murine AD models ,66. In pGenetic variants of Lrp6 have been investigated for being a risk factor for late-onset AD. For example, the Ile-1062-Val variant (exon 14) of Lrp6, which reduces the activity of Wnt/β-catenin signaling, appears to be a genetic risk factor for AD . MoreoveIn addition to the protein components of the Wnt pathways, the lipid membrane microenvironment also plays key roles in AD. For example, cholesterol content and distribution are associated with Aβ production and cell dysfunction in AD . Aβ appeAlthough Aβ accumulation and abnormal Tau protein accumulation are the most widely accepted mechanisms for AD, they are insufficient to explain the disease mechanism and to target AD therapeutically ,61. For Parkinson’s disease (PD) is the second most common neurodegenerative disease, following AD, with an age-dependent prevalence of 1-4% ,97,98. PDrosophila model of PD has been found to significantly reduce disease-related abnormalities, such as impaired flight ability, by inhibiting autophagy and apoptosis and restoring mitochondrial function [The pesticides paraquat and maneb, which interfere with mitochondrial function and cause toxicity via oxidative stress, have been shown to cause neurotoxicity in the dopaminergic system and, thus, increase the risk of PD ,105. Botfunction . These dfunction .Properties of the membrane lipid environment can affect the prognosis of PD. The familial PD-linked proteins α-synuclein, LRRK2, parkin, and DJ-1 have been demonstrated to be associated with the lipid rafts, strongly suggesting that lipid rafts are involved in the pathogenesis of PD ,111. E3 Schizophrenia (SZ) is a chronic brain disorder and severe mental disorder that is characterized by aberrant neural lamination and orientation in the hippocampus. Patients with SZ interpret reality abnormally, and are characterized by extremely disorganized and abnormal motor activity, behavioral variability, delusions, disorganized thinking, and hallucination ,117. NeuDuring the development of SZ, lipid homeostasis in the CNS also affects the fatty acid content in lipid rafts. Dietary supplementation of unsaturated omega-3 fatty acids causes their incorporation into the neural cell plasma membrane, increases membrane permeability, and modifies the organization of the lipid rafts ,139. TheWnt signaling is a major regulator of the development and growth of various tissues and organs involved in bodily metabolism, relating it with a range of metabolic diseases including diabetes, obesity, NAFLD, and NASH, which will be discussed here.Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, which begins with isolated steatosis and advances to nonalcoholic steatohepatitis (NASH), steatofibrosis, and cirrhosis. TCF7L2/TCF4 as a susceptibility gene for T2DM, and associated various TCF7L polymorphisms with a significantly higher risk of developing T2DM [Several in vitro and in vivo studies have shown that the components of the Wnt signaling pathway are involved in β-cell proliferation, insulin secretion, and lipid metabolism ,142,143.ing T2DM ,148,149.ing T2DM . Wnt siging T2DM . Furthering T2DM . The Wnting T2DM ,152. SOST gene in osteocytes, and was found to be expressed at higher levels in T2DM subjects than in controls [Due to the potential functions of the Wnt/β-catenin signaling pathway on bone development and remodeling, its dysregulation in T2DM makes patients more susceptible to bone complications. Sclerostin, which antagonizes the Wnt/β-catenin pathway by binding to Lrp5/6, is a small protein expressed by the controls ,154. Intcontrols . Vascular calcification is one of the most common complications in patients with T2DM. miR-128-3p accelerates cardiovascular calcification and insulin resistance in T2DM rats by targeting the pancreatic islet endocrine cell marker ISL-1 and activation of the Wnt pathway . UpregulThe Wnt/β-catenin signaling pathway plays a vital role in adipose tissue lipogenesis and adipocyte metabolism—particularly under obesogenic conditions ,158. A cR611C mutation exhibit both steatohepatitis and steatofibrosis features associated with NAFLD [R611C mice was efficiently remedied by administration of Wnt3a. Lrp6 knockdown also stimulated the non-canonical Wnt proteins RhoA (Ras homolog family member A) and ROCK2 (Rho-associated protein kinase), as well as their phosphorylated forms. Thus, the Lrp6 and non-canonical Wnt pathways are likely to be important therapeutic targets against NAFLD and NASH. The role of Lrp6 has also been evaluated in another study, where miR-21 was found to inhibit Lrp6 expression and Wnt β-catenin signaling activity, and enhanced the expression of critical lipid metabolic enzymes [The liver has critical functions in the control of metabolic pathways such as glucose metabolism and fatty acid metabolism . The Wntth NAFLD . Impaire enzymes . These rCompositions of phospholipid fatty acids have been found to alter in the cell membranes of patients with T2DM, obesity, metabolic syndrome, or NAFLD ,171,172.Cholesterol appears to be another major actor that plays a role in the development of NASH. Dysregulation of hepatic cholesterol homeostasis causes accumulation of hepatic free cholesterol (FC) and oxidized low-density lipoprotein (oxLDL) in NAFLD and NASH ,187,188.The Wnt signaling pathways are essential for many cellular events that take place in development, homeostasis, and regeneration. Being the initiator of Wnt–receptor complex formation that activates the signaling pathway, the plasma membrane plays an essential role in the regulation of Wnt signaling. Signaling initiation and regulation strongly depend on the content and organization of the membrane. In pathological processes, including brain disorders and metabolic diseases, changes occur in the composition of membrane lipids and proteins. While the Wnt signaling pathway has been relatively better characterized in AD and PD with respect to the content and organization of the plasma membrane domains, there exists limited knowledge concerning this issue in SZ. Therefore, further studies are required in order to clarify the role of the Wnt signaling pathway in SZ in the context of plasma membrane organization. Understanding the molecular mechanism of the Wnt signaling pathway in the context of plasma membrane organization will contribute to the development of new therapeutic strategies for the diseases in which the Wnt signaling pathway is dysregulated.


Uric acid (UA) is the final product of the catabolism of endogenous and exogenous purine nucleotides. While its association with articular gout and kidney disease has been known for a long time, new data have demonstrated that UA is also related to cardiovascular (CV) diseases. UA has been identified as a significant determinant of many different outcomes, such as all-cause and CV mortality, and also of CV events (mainly Acute Coronary Syndromes (ACS) and even strokes). Furthermore, UA has been related to the development of Heart Failure, and to a higher mortality in decompensated patients, as well as to the onset of atrial fibrillation. After a brief introduction on the general role of UA in CV disorders, this review will be focused on UA’s relationship with CV outcomes, as well as on the specific features of patients with ACS and Chronic Coronary Syndrome. Finally, two issues which remain open will be discussed: the first is about the identification of a CV UA cut-off value, while the second concerns the possibility that the pharmacological reduction of UA is able to lower the incidence of CV events. Uric Acid (UA) is the final product of the catabolism of purine nucleotides from endogenous (cellular nucleoproteins) and exogenous origins . Its biosynthesis, which principally involves the liver, also includes the gut, muscles and kidneys; urinary excretion is the main mechanism of UA elimination, while a small percentage thereof is removed by the intestine. At a pH of 7.4, the solubility limit of plasma UA is 6.8 mg/dL. Beyond this level, the conditions for urate crystal precipitation are created. Conditions which may raise UA levels include the increased production which occurs with a purine-rich diet, tumor lysis syndrome or specific drugs (chemotherapy and pyrazinamide), and also with a decrease in UA excretion, mainly in renal diseases. Genetics could also be a cause for hyperuricemia, as it happens in the gain of function of the enzyme phosphoribosyl-pyrophosphate synthetase, or in the deficit of hypoxantine-guanine phosphoribosyltransferase  .The association of UA with articular gout and kidney disease has been known for a long time, while new data has demonstrated that UA is also related to cardiovascular (CV) diseases.  In fact,Hyperuricemia represents an epidemiological problem, especially if CV comorbidities are present. Its prevalence ranges from 6% in healthy subjects  to 14% iThe connection between UA and CV events was demonstrated for the first time in 1967 by Kannel et al.  in the FThe mechanisms by which UA could determine CV events have not been definitively identified. However, UA certainly acts at multiple levels, as shown in First, the oxidative stress determined by the two final biochemical reactions is involved: during the conversion of hypoxanthine into xanthine (and hence into UA) determined by the xanthine oxidase enzyme, the generated superoxide anions increase oxidative stress, a well-known atherosclerotic risk factor ,16. In aOxidative stress represents a fundamental pathway in diseases related to hyperuricemia (hypertension and Diabetes Mellitus—DM), and in the development of heart and vessels organ damage. In fact, UA has been linked to the development of arterial hypertension , DM 19]19], and One of the most important mechanisms through which UA is probably related to CV events is renal damage, a well-known CV risk factor. UA can affect kidneys by depositing crystals in renal tubules during hyperuricosuria , resultiFinally, UA also appears to be linked to heart and vessel damages. In particular, the relationship with pulse wave velocity, which is the most widely used measurement of arterial stiffness, suggests a possible association between UA and changes in vascular structure and function .Another relevant aspect in the relation between UA and CV diseases is the role of gender. Some studies describe a link with target organ damage only in females , while CAs was already mentioned, hyperuricemia appears to be associated with fatal and non-fatal ACS in the general population . In patiIn particular, many studies found an association between UA levels at admission and specific HF-related issues, such as the Killip class, the use of intra-aortic balloon pump and cardiogenic shock, and a reduced left ventricular ejection fraction at admission ,36. ThisSome studies identify UA as a determinant of a more severe coronary artery involvement, a larger infarct size , a greatIf the assumption that UA acutely increases during hospitalization due to secondary hemodynamic effects is true, a decrease in its values from admission to discharge should be expected. However, data on longitudinal UA changes in ACS subjects are still lacking.Hyperuricemia is a significant epidemiological problem in CCS, and has been strongly connected to CV mortality and CV events in this specific subgroup of patients ,45,46,47Gender could be another factor influencing the UA–CAD association. Only two studies carried out a separate analysis in males and females, finding a connection only in the latter . In addiThe commonly-used cut-offs of 6 mg/dL in women and 7 mg/dL in men were established on evidence regarding gouty patients, rather than CV events in asymptomatic hyperuricemia. These thresholds are based on the UA saturation point (6.8 mg/dl at a pH of 7.4) which determines its precipitation in joints and kidneys, leading to the classic form of gout. However, previous evidence suggests that UA could act negatively on the CV system even at lower serum levels , as crysDespite the large number of published studies, the identification of a CV UA cut-off value is still a matter of discussion. Among others, recently published results from an Italian multicenter, retrospective, observational cohort study brought new light on the CV cut-off question. The URRAH project  entailed data collection on outpatients (mainly hypertensives) and the general population, with a total of 23,475 subjects and a follow-up period of 20 years. Regarding all-cause mortality, a threshold of 4.7 mg/dL was detected, while 5.6 mg/dL emerged as the most suitable cut-off for CV mortality . In bothAnother interesting point is the identification, in some studies, of a J-curve in the relationship between UA levels and CV events, meaning not only that high levels of UA raise the risk but also that too-low values could be harmful ,62. For Currently, the therapies for the reduction of UA levels are exclusively recommended in patients with hyperuricemia associated with gouty arthritis or gouty nephropathy; these include xanthine oxidase inhibitors (Allopurinol and Febuxostat) and uricosurics (Probenecid and Lenisurad). Xanthine oxidase inhibitors were the first registered drugs, and Febuxostat showed the greatest inhibiting effectiveness with a complete selectivity for xanthine oxidase, while allopurinol also works on other enzymes involved in purine metabolism. Instead, Probenecid and Lenisurad increase the urinary excretion of UA acting on a specific renal transporter. Many other molecules are under development , but theSome observational studies reported a decrease of CV events in patients treated with hypouricemic drugs , but, asTwo RCTs limited to individuals with gout have been published, showing opposite results. The CARES (Cardiovascular safety of Febuxostat and Allopurinol in patients with gout and cardiovascular comorbidities) study randomized 6190 patients with gout and previous CV events to Allopurinol vs. Febuxostat . The subFor this latter group, one study is on-going and another one has been already published. The FREED  trial  comparedUnfortunately, no information about the efficacy of UA reduction in primary CV prevention is available because of the lack of studies on asymptomatic hyperuricemia in individuals without prior CV events.Eventually, regarding ACS, one specific but small and non-randomized study is available ; it enroAlthough many findings have been published in favor of a role of UA in CV diseases (particularly in ACS and CCS), several points remain not completely understood in this complicated relationship. From a pathophysiological point of view, the question of whether hyperuricemia contributes directly to the genesis of ACS and CCS, or if it is just an innocent bystander determined by an increased catabolism in ischemic myocardium is still a matter of debate. Furthermore, the lack of an unequivocally accepted UA CV cut-off does not allow us to define a clear threshold of CV events’ risk and their fatality. While waiting for further results on this issue, a more suitable cut-off must be chosen on an individual basis, strongly depending on the patients’ CV risk and previous CV events. Finally, there is currently no strong evidence about a certain benefit deriving from a pharmacological treatment of hyperuricemia in terms of the reduction of CV morbidity and mortality. The on-going ALL-HEART trial will provide us with an important answer, by determining whether allopurinol reduces CV events in patients with CCS, and it could perhaps change the role of UA in secondary CV prevention strategies. In conclusion, more studies should be performed in order to clarify the involvement of this molecule in the spectrum of CV disease and its possible role as a target in CV prevention strategies.


The optimal management of neonatal post-hemorrhagic hydrocephalus (PHH) is still debated, though several treatment options have been proposed. In the last years, ventriculosubgaleal shunt (VSgS) and neuroendosdcopic lavage (NEL) have been proposed to overcome the drawbacks of more traditional options, such as external ventricular drainage and ventricular access device.We retrospectively reviewed neonates affected by PHH treated at our institution since September 2012 to September 2020. Until 2017 patients received VSgS as initial treatment. After the introduction of NEL, this treatment option was offered to patients with large intraventricular clots. After NEL, VSgS was always placed. Primary VSgS was reserved to patients without significant intraventricular clots and critically ill patients that could not be transferred to the operating room and undergo a longer surgery.We collected 63 babies  with mean gestational age of 27.8 ± 3.8SD weeks (range 23–38.5 weeks) and mean birthweight of 1199.7 ± 690.6 SD grams (range 500–3320 g). In 6 patients, hemorrhage occurred in the third trimester of gestation, while in the remaining cases hemorrhage complicated prematurity. This group included 37 inborn and 26 outborn babies. Intraventricular hemorrhage was classified as low grade  in 7 cases, while in the remaining cases the grade of hemorrhage was III to IV. Mean age at first neurosurgical procedure was 32.2 ± 3.6SD weeks (range 25.4–40 weeks). Death due to prematurity occurred in 5 patients. First-line treatment was VSgS in 49 patients and NEL in the remaining 14 cases. Mean longevity of VSgS was 30.3 days (range 10–97 days) in patients finally requiring an additional treatment of hydrocephalus. Thirty-two patients required one to three redo VSgS. Interval from initial treatment to permanent shunt ranged from 14 to 312 days (mean 70.9 days). CSF infection was observed in 5 patients (7.9%). Shunt dependency was observed in 51 out of 58 surviving patients, while 7 cases remained shunt-free at the last follow-up. Multiloculated hydrocephalus was observed in 14 cases. Among these, only one patient initially received NEL and was complicated by isolated trapped temporal horn.VSgS and NEL are two effective treatment options in the management of PHH. Both procedures should be part of the neurosurgical armamentarium to deal with PHH, since they offer specific advantages in selected patients. A treatment algorithm combining these two options may reduce the infectious risk and the risk of multiloculated hydrocephalus. Neonatal post-hemorrhagic hydrocephalus (PHH) is a complication of intraventricular hemorrhage (IVH) in preterm babies  or, lessIn this context, ventriculo subgaleal shunt (VSgS) has emerged in the last years as a solution reducing the infectious risk and allowing to control hydrocephalus until the patient reaches an adequate body weight and the CSF clarifies . More reWe sought to develop a treatment algorithm that implements both procedures, focusing our attention on the selection of patient candidate to VSgS with or without NEL.We retrospectively collected neonatal patients affected by PHH treated at the Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy, since September 2012 to September 2020. Charts were reviewed for demographic, clinical, and surgical data. Until 2017, all patients were initially treated by VSgS. After the introduction of NEL, indication to VSgS versus NEL+VSgS was carefully based on the preoperative US evaluation and clinical conditions of the baby. In fact, VSgS was indicated in cases with small intraventricular clots and preserved communication between lateral and third ventricles, warranting drainage of the ventricular system by a single ventricular catheter. On the other hand, NEL+VSgS was indicated in case of large intraventricular clots, aiming to prevent multiloculated hydrocephalus. Main contraindication to NEL+VSgS was represented by critical general conditions requiring treatment in the intensive care unit, without any possibility to transfer the patient to the surgical theater.Our surgical technique for placement of VSgS has been extensively described elsewhere . The proAfter careful disinfection and sterile draping, an L-shaped incision is performed in the right pre-coronaric area. This incision warrants adequate skin covering over the device, also at the time of redo VSgS, and can be easily converted in a C-shaped incision at the time of shunt conversion. A small burr hole is made using a No. 20 scalpel blade on the anterior angle of the bregmatic fontanelle, followed by dural opening and cortical coagulation. A large subgaleal pouch is harvested before ventriculostomy with blunt dissection of the subgaleal space in the ipsilateral parietal region. The device is prepared using an antibiotic-impregnated catheter  with the interposition of a right-angle connector. The distal end is left open with additional holes in the ending segment of the tube Fig. . After vThe surgical technique has been extensively described in the literature , 7. In tPostoperatively, all patients are transferred to the neonatal intensive care unit. Lateral positioning contralaterally to the subgaleal pouch is recommended, thus avoiding compression of the pouch. Clinical examination monitoring the tension of the subgaleal pouch and the head circumference is performed daily, while wound care as well as transfontanellar US are accomplished every third day. If clinical signs of active hydrocephalus reoccur, due to VSgS malfunction , conversion to permanent ventriculo-peritoneal shunt is considered if general clinical conditions are stable, patient’s weight is over 2000 g, and hemorrhage is solved on US/MR. In case of previous abdominal surgery, conversion to ventriculo-atrial shunt is indicated and it is performed if the diameter of the jugular vein is adequate and patient’s weight is over 5000 g.Revision of VSgS is performed if patient is not suitable for conversion into permanent shunt. If VSgS is revised, the device is replaced exploiting the same frontal access and harvesting a new subgaleal pouch on the opposite side or in a site that was not dissected before. In case of conversion to permanent shunt, the shunt is placed exploiting the existing frontal access. Finally, patient may be discharged with working VSgS and monitored into outpatient clinic every other week with transfontanellar US. In case of collapsed pouch without recurrent hydrocephalus, VSgS is surgically removed, thus making the patient shunt-free.We collected 63 babies  with mean gestational age of 27.8 ± 3.8SD weeks (range 23–38.5 weeks) and mean birthweight of 1199.7 ± 690.6SD grams (range 500–3320 g). In 6 patients, hemorrhage occurred in the third trimester of gestation, while in the remaining cases hemorrhage complicated prematurity. This group included 37 inborn and 26 outborn babies. Intraventricular hemorrhage was classified as low grade  in 7 cases, while in the remaining cases the grade of hemorrhage was III to IV. Mean age at first neurosurgical procedure was 32.2 ± 3.6SD weeks (range 25.4–40 weeks). Death due to prematurity and related complications was registered in 5 patients.First-line treatment was VSgS in 49 patients and NEL in the remaining 14 cases. Mean time of VSgS was 30.3 days, ranging from 10 to 97 days in patients finally requiring an additional treatment of hydrocephalus. Thirty-two patients required one to three redo VSgS. Additional treatments in patients receiving VSgS as first treatment were NEL in 4 cases, EVD in 9 cases, and other endoscopic procedures for fenestration of intraventricular septa in 3 cases. One patient was complicated by subdural hygroma, requiring conversion of the VSgS into a subduro-subgaleal shunt Fig. . One patTime from initial treatment to permanent shunt ranged from 14 to 312 days (mean 70.9 days). Interestingly, 20 patients were discharged with VSgS.CSF infection was observed in 5 patients (7.9%). In particular, infection finally complicated CSF leak in three cases who underwent several procedures for treatment of multiloculated hydrocephalus before the introduction of NEL Fig. . In the Shunt dependency was observed in most of the cases with conversion to ventriculo-peritoneal shunt in 43 cases and ventriculo-atrial shunt in 8 cases. Seven cases remained shunt-free at the latest follow-up; two of them received ETV at the time of VSgS removal.Multiloculated hydrocephalus was observed in 14 cases, requiring isolated aqueductoplasty in 4 cases, septostomy in 2 cases, fenestration of trapped temporal horn in 2 cases, and fenestration of multiple intraventricular septa/cysts in the 6 remaining cases. Among these, only one patient initially received NEL and was complicated by isolated trapped temporal horn.With regard to the implementation of NEL in our treatment algorithm, thirty-nine patients were treated in the first part of our experience (VSgS) and 24 after the introduction of the new protocol (VSgS ± NEL). Results are presented in Table The treatment of neonatal PHH is still challenging due to the fragility of patients and other critical factors. Indeed, features of either the patient, namely prematurity and low weight, and the CSF, such as the blood clots and high protein concentration, and the receiving site of extrathecal CSF shunting, that are necrotizing enterocolitis for the abdomen and inadequate caliber of jugular vein in case of ventriculo-atrial shunt, require transient treatment of hydrocephalus, thus delaying the timing of permanent shunt until the previous conditions are solved.External ventricular drainage (EVD) may be inserted at bedside, theoretically warrants continuous CSF drainage, and can be easily removed . These aOn these grounds, we reserve EVD to patients with infection of CSF or blood, who could not receive VSgS.Ventricular access device (VAD) partly overcomes limits of EVD , 18, butThese considerations prompted us to adopt VSgS in the management of PHH. Indeed, this treatment option proved to be effective and safe. Main advantages of VsgS are avoiding the risk of electrolyte imbalance secondary to CSF drainage, the easiness to manage the patient by the nursing and medical staff after adequate education, and the possibility to discharge the patient to home before permanent treatment of hydrocephalus. Furthermore, the length of the procedure is usually less than 15 min and could be performed at the bedside.Infectious risk seems lower compared to other treatment options, namely EVD and VAD. It is of worth to note that series of VSgS reporting no infection include less than 20 patients , 20, whiOther complications are hemorrhage, due to abrupt drainage of ventricular system, and subdural hygroma hematoma.Skin dehiscence with device exposure and CSF leak may be secondary to loose skin. Additionally, VSgS shares mechanical complications, such as obstruction and migration, with other shunt devices .Technical aspects of VSgS are not standardized through the literature, in particular concerning the use of valve versus valveless tube and the use of reservoir. The use of valve may reduce the risk of abrupt drainage of the ventricular system and related complications, but it is associated to a major risk of obstruction by blood clot and collapsed subgaleal pouch. On these grounds, daily pumping is recommended by centers using VSgS with valve , 24.Additionally, the management of VSgS when subgaleal pouch is satured varies across the literature. In the majority of experiences, tapping of the pouch or of the reservoir is performed to extend the life of the VSgS. This somehow converts the VSgS into a ventricular access device and may affect the comparison of morbidity between the two options .In our experience, we designed a bundle exploiting antibiotic-impregnated catheter and avoiding to tap the device or the pouch aiming to reduce infectious risk. Indeed, infection is the only factor, along with entity of brain damage by the initial hemorrhage, affecting the neurocognitive outcome of these patients . AdditioOur management protocol of VSgS warranted an acceptable risk of infection, though this risk is ineluctable as the device and CSF could be infected secondarily to blood sepsis.With the present no-tapping policy, the mean life expectancy of VSgS is about 32 days and it could be not compared to other experiences using daily tapping to extend the life of VSgS after saturation of the subgaleal pouch.Other complications, secondary to abrupt drainage of the ventricular system, such as subdural hygroma and intraventricular hemorrhage occurred in the initial phase of this experience.Some surgical nuances, as accurate choice of the side of ventriculostomy avoiding area with very thin brain parenchymal thickness and continuous irrigation of the subgaleal pouch during skin closure, helped to prevent these complications.This retrospective analysis revealed three cases complicated by primary CSF infection without general sepsis before the introduction of NEL. Risk factors were several surgical and endoscopic procedures for multiloculated hydrocephalus and CSF leak. Indeed, the presence of large intraventricular clots favored a bilateral entrapment of temporal horns.On these grounds, we implemented NEL in our management algorithm of PHH to prevent multiloculated complications and related morbidity Fig. , as suppThese treatment options have been proposed as alternative options by different neurosurgical centers. However, VSgS and NEL may have different indications and should be both available in the surgical armamentarium of pediatric neurosurgeons.VSgS may be placed in the intensive care unit , thus reOn the other side, NEL should be performed in the surgical theater and it reduces the risk of multiloculated hydrocephalus in patients with large intraventricular clots Fig. . This trLiterature data also show that NEL could significantly reduce the rate of shunt dependency . HoweverIn conclusion, this study represents the first institutional experience combining VSgS and NEL in the management of PHH. Although longer follow-up is required to study the neurocognitive outcome of these patients , these dVSgS and NEL are two effective treatment options in the management of PHH. Both procedures should be part of the neurosurgical armamentarium to deal with PHH, since they offer specific advantages in selected patients. A treatment algorithm combining these two options may reduce the infectious risk and the risk of multiloculated hydrocephalus.