Dynamic changes in the molecular orientation and interphase structures of SiO2/SMPU during a shape memory cycle were then discussed according to the small angle X-ray scattering theory, Guinier’s law, Porod approximation, and fractal dimension theorem. In this paper, a dynamic mechanical analyzer (DMA) helped to determine the glass transition start temperature (Tg) by taking the onset point of the sigmoidal change in the storage modulus, while transition temperature (Ttrans) was defined by the peak of tan δ, then the test and the calculated results indicated that the Tg of SiO2/SMPU was 50.4 °C, and the Ttrans of SiO2/SMPU was 72.18 °C. SiO2/SMPU showed good shape memory performance. The programmed SiO2/SMPU showed quite obvious microphase separation and molecular orientation. Large-size sheets and long-period structures were formed in the programmed SiO2/SMPU, which increases the electron density difference. Furthermore, some hard segments had been rearranged, and their gyration radii decreased. In addition, several defects formed at the interfaces of SiO2/SMPU, which caused the generation of space charges, thus leading to local electron density fluctuations. The blurred interphase structure and the intermediate layer formed in the programmed SiO2/SMPU and there was evident crystal damage and chemical bond breakage in the recovered SiO2/SMPU. Finally, the original and recovered SiO2/SMPU samples belong to the surface fractal system, but the programmed sample belongs to the mass fractal and reforms two-phase structures. This study provides an insight into the shape memory mechanism of the SiO2/SMPU composite.In order to further understand the shape memory mechanism of a silicon dioxide/shape memory polyurethane (SiO A shape memory polymer (SMP), as a kind of smart material, can restore its original shape due to heat, magnetism, photo, and other external stimulation , which i2), and so on  − q2 based on the SAXS test results of the original, programmed, and recovered SiO2/SMPU samples [2 particles in the SiO2/SMPU, which led to electron density fluctuations at the above interfaces. The interphase interaction between molecular chains of the SMPU matrix and SiO2 particles is the main cause of a positive deviation [ samples . It can eviation .2 particles. The defects between them further result in the generation of space charges, which affects the electron density [2 particles and the SMPU matrix, is not a stepwise gentle transition process, but a shielding process with a “jumping” property, which causes the electrons in the interface phase to be disturbed by the charge force and localized density fluctuations occur.On one hand, there are many formed defects at the interfaces between the hard and soft segments as well as between the SMPU matrix and SiO density . On the 2/SMPU samples. This minor difference was caused by the crystal damages and chemical bond breakages that appeared in the recovered SiO2/SMPU during the shape memory cycle. Furthermore, the tangled structures between the soft and hard segments in the SMPU matrix as well as between the SiO2 particles and SMPU matrix were changed and could not be restored to their original states, leading to differences in electron density.Additionally, 2/SMPU sample, followed by the appearance of a positive deviation. This deviation trend was consistent with those of the original and recovered SiO2/SMPU samples. The occurrence of a negative deviation indicates that the blurred interface structure appeared in some states of the programmed SiO2/SMPU sample, and an intermediate layer appeared [A short negative deviation transition state appeared on the Porod curve of the programmed SiOappeared .2 particles are simultaneously moved in order to rearrange during the programming process. Additionally, some SiO2 particles are extruded with the pore structures of the porous SMPU being damaged. SiO2 particles with larger surface effects absorb some media, which reduces its surface energy, forming an interface layer on the SiO2 particle surface, and weakening the scattering intensity. It is demonstrated that the thermodynamic shape memory cycle will inevitably bring some microscopic damages to the SiO2/SMPU composite.The appearance of this negative deviation was due to the molecular chains of the soft segments being oriented, while the hard segments and SiO2/SMPU sample presented a distinct linearity in the entire wave vector region, which showed a self-similarity. The slope values of the double-log curves were 3.71 and 3.69, respectively, and belong to the surface fractal system [2/SMPU sample was slightly larger than that of the recovered sample, the surface of the original SiO2/SMPU sample was slightly rougher than that of the recovered SiO2/SMPU sample, as shown in 2 particles moving and filling in the SMPU matrix pores during the orientation of molecular chains in soft segments. Therefore, some physical entanglement structures were formed between the SiO2 particles and hard segments, which resulted in some damage to the chemical bonds and crystal structures.As shown in l system . As the 2/SMPU showed the non-surface fractal phenomenon and fractal transition stage in the middle wave vector region [2 particles were squeezed, which led to the damage of some crystal lattices and chemical bonds, resulting in forming some microcrystal domains.In r region , which w2/SMPU samples, the entire transition state shifted with the appearance of some large particles. These particles are derived from the physical connection between the SiO2 particles and molecular chains in hard and soft segments. The formation of hydrogen bonds among them is one of the most important reasons why the programmed SiO2/SMPU is difficult to completely restore to its original shape.When compared to the double-log curves of the original and recovered SiO2/SMPU, the slope value of the double-log curve was 2.27, which belongs to the mass fractal system. It was further confirmed that as molecular chains of reversible soft segments are stretched, and hard segments and SiO2 particles are also moved. As a result, the tightness degree of the SiO2/SMPU composite increased.In the fractal state of the programmed SiO2/SMPU composite was prepared, and its Ttrans and shape memory behaviors of SiO2/SMPU were characterized and the dynamic changes in molecular orientation and interphase structures during a shape memory cycle were discussed. Main conclusions were obtained as follows.In this study, a SiOTg of prepared SiO2/SMPU was 50.4 °C, and the Ttrans was 72.18 °C. The molecular chain begins to move and the mechanical properties of SiO2/SMPU begin to change at Tg, and it has the best shape memory effect and mechanical properties at Ttrans.The 2 have little influence on Pure SMPU, but after a shape memory cycle, SiO2/SMPU had microscopic loss and segment damage after the recovery process, but still had a good shape memory effect, which was demonstrated in the shape memory test. After a whole cycle, the Rf and Rr of SiO2/SMPU could still reach above 97%.The XRD curves showed that the addition of SiO2/SMPU experience a process from isotropy to anisotropy, and back to isotropy during a shape memory cycle. The different scattering intensities in vertical and horizontal direction manifest that the programmed SiO2/SMPU has an obvious microphase separation and molecular orientation.The molecular chains of SiO2 particles moved with the extension of the molecular chain moving along the stretching direction, and partially filled in the pores caused by the intense reaction; some large-size sheet structures were also formed during the programming process. This further caused the change in electron density between the crystalline and amorphous regions in the SMPU matrix. According to the SAXS Lorentz correction, some long-period structure is formed in the programmed SiO2/SMPU, revealing the shape memory mechanism of SiO2/SMPU.The SiO2/SMPU were affected by the molecular chain movement to deviate from the spherical shape, and were rearranged along the stretching direction. SiO2 particles influence the shape symmetry of a hard segment, and the gyration radius of the hard segment scatterer was increased after the programming process of SiO2/SMPU.The SAXS profiles of the scattering curves suggest that some hard segments in the programmed SiO2 particles. These defects produced a heterogeneous distribution of positive and negative charges at the interfaces, leading to local electron density fluctuations.The Porod curves showed that some defects formed at the interfaces between hard and soft segments as well as between Pure SMPU and SiO2 particles moved along the stretching direction of molecular chains of soft segments in the programming process, after that, the blurred interphase structure and intermediate layer formed in the programmed SiO2/SMPU. During the whole cycle of shape memory, there were some crystal damage and chemical bond breakages in the recovered SiO2/SMPU.The hard segments and SiO2/SMPU samples showed a self-similarity, which is seen as a surface fractal. The programmed SiO2/SMPU showed a non-fractal phenomenon and fractal transition region, which is seen as mass fractal. This further confirms that hard segments and SiO2 particles are moved together, and reform the two-phase structure in programmed SiO2/SMPU.Calculated fractal dimensions of original and recovered SiO


The noncoding control regions (NCCRs) of the sequenced viral strains were rearranged. Conclusions: HPyVs other than JCPyV were found in the CSF of patients affected with different neurological diseases, probably as bystanders, rather than etiological agents of the disease. However, the fact that they can be latent in the CNS should be considered, especially in immunosuppressed patients.Background: Central nervous system (CNS) infections by human polyomaviruses (HPyVs), with the exception of JC (JCPyV), have been poorly studied. Methods: In total, 234 cerebrospinal fluid (CSF) samples were collected from patients affected with neurological disorders. DNA was isolated and subjected to quantitative real-time PCR (Q-PCR) for the detection of six HPyVs: JCPyV, BKPyV, Merkel cell PyV (MCPyV), HPyV6, HPyV7, and HPyV9. Where possible, the molecular characterization of the viral strains was carried out by nested PCR and automated sequencing. Results: JCPyV was detected in 3/234 (1.3%), BKPyV in 15/234 (6.4%), MCPyV in 22/234 (9.4%), and HPyV6 in 1/234 (0.4%) CSF samples. JCPyV was detected at the highest ( Polyomaviridae family comprises small, naked, DNA viruses, sharing the same structure. In particular, they have a circular DNA genome of approximately 5 kb divided into a region encoding the early, functional proteins, small and large tumor antigens, a region encoding the late, structural proteins, VP1 and VP2, and a noncoding control region (NCCR), containing the origin of replication and the promoters. The VP1 region is prone to point mutations and is useful for viral genotyping, while the NCCR is a hypervariable region that is susceptible to mutation and heterogeneous rearrangement, leading to pathogenic consequences [The Polyomaviruses infect a broad spectrum of hosts, including humans. Since 1971, at least 14 human polyomaviruses (HPyV) have been identified in different biological specimens  ,13,14,15So far, a link with human diseases has been established for six of the identified HPyVs. JC polyomavirus (JCPyV) is the etiological agent of the demyelinating disease of the central nervous system (CNS), progressive multifocal leukoencephalopathy (PML) ; BK polyAs stated, the CNS tropism of JCPyV is very well defined, and BKPyV is occasionally found in both brain tissue and the cerebrospinal fluid (CSF) ,24. In cThus, we decided to analyze the presence of MCPyV and HPyV6, 7, and 9, in addition to JCPyV and BKPyV, genomes in CSF samples collected from a large cohort of patients affected by different neurological diseases.The final aim was to assess the possible relevance of other HPyVs, besides JCPyV and BKPyV, in neurological disorders mostly not related to other microbial agents.In this retrospective, observational study, CSF leftovers after routine laboratory tests were collected and stored at −70 °C from 234 patients, 175 of whom were admitted at the University Hospital Tor Vergata, Rome, and 59 admitted at the Neurological Institute Mondino, Pavia, Italy, from March 2016 to November 2017. Among this group, we included patients with meningoencephalitis  and patients with other neurological conditions: peripheral neuropathies , cognitive disorders , multiple sclerosis , brain tumor , epilepsy , confused state  hydrocephalus , migraine , myelitis , and mood disorders . CSF collection was performed at the time of the first diagnosis. Paired sera were also collected for the 59 cases admitted at the Neurological Institute Mondino. Other demographic data are reported in All CSF samples were subjected to testing for the presence of human herpesviruses (herpes simplex virus-1 and -2 (HSV-1 and -2)), varicella zoster virus (VZV), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), and enteroviruses for diagnostic purposes. Four CSF samples were also subjected to HIV testing, because of signs of cognitive deterioration in HIV+ patients.The study was approved by the local Ethics Committee Fondazione PTV Policlinico Tor Vergata Protocol no. 211/17, date 08 January 2018 and all patients signed a free and informed consent form during clinician examination.DNA isolation was performed from 150 μL of CSF and from the available sera using the spin-column technique  according to the manufacturer’s instructions. All laboratory procedures were carried out under stringent conditions to avoid contamination, as follows: samples were processed individually; DNA isolation was performed one sample at a time in a clean, dust-free area with sterile and disposable materials, with a change of gloves for each sample.5–10 copies/μL) containing whole genomes of JCPyV, BKPyV, HPyV6, and HPyV7  and partial genomes of MCPyV, kindly provided by H. Feng, and HPyV9, kindly provided by M.C.W. Feltkamp. The limit of detection was 2 copies/reaction for JCPyV and BKPyV and 10 copies/reaction for MCPyV, HPyV6, HPyV7, and HPyV9.HPyV Q-PCR assays were conducted with DNA isolated from the CSF. The Q-PCR protocols for the detection of JCPyV, BKPyV, MCPyV, HPyV6, HPyV7, and HPyV9 DNA have been previously published ,32,33,34Only the serum samples paired with positive CSF were tested for the presence of HPyVs. Thus, Q-PCR for the detection of MCPyV and/or BKPyV was conducted on 17 serum samples paired with 17 MCPyV- or BKPyV-positive CSF samples.Although the CSF was spun before DNA isolation, we hypothesized that some cells may remain. To determine the percentage of infected cells, a concomitant Q-PCR assay targeting the β-globin gene was performed on the HPyV-positive samples. The β-globin gene was amplified using a previously published primer set and the thermal cycling conditions . The reshttps://blast.ncbi.nlm.nih.gov) were used to determine the sequence homology, as previously described by Agostini et al. [Molecular characterization of the positive strains was possible in only some cases because of a lack of specimens. CSF samples with positive results for the presence of BKPyV and/or JCPyV genomes were molecularly characterized using nested PCR for the VP1 genotyping fragment and NCCR, as previously described . Additioi et al.  for JCPyi et al.  and Jensi et al.  for JCPyi et al. . The MCP2 test and the Fisher exact test were used to evaluate the significance of the association among HPyV infection, HPyV load, and HPyV infection and neurological disease; p < 0.05 was considered significant.The χp < 0.05). Additionally, three CSF samples were positive for the presence of both BKPyV and MCPyV genomes, as reported in Overall, HPyV genomes were detected in 41/234 (17.5%) CSF samples. Of the six searched HPyVs, JCPyV was detected in 3/234 (1.3%), BKPyV in 15/234 (6.4%), MCPyV in 22/234 (9.4%), and HPyV6 in 1/234 (0.4%) CSF samples. BKPyV and MCPyV were significantly more frequently detected than JCPyV  mean load , followed by BKPyV , MCPyV , and HPyV-6 (3.3 × 104 copies/mL).The cycle threshold (CT) for every positive sample is reported in Additionally, the percentages of infected cells were evaluated: JCPyV infected the highest mean percentage of cells , followed by BKPyV , and MCPyV .The patients infected with HPyVs were affected by meningoencephalitis (25), peripheral neuropathy/polyradiculoneuropathy (5), PML (3), Sjogren syndrome (1), cognitive disorder (1), migraine (1), myeloproliferative disease (1), angioma (1), multiple sclerosis (1), mood disorders (1), and brain tumor (1), as seen in Molecular characterization of the HPyV-positive strains was not performed for all the positive CSF samples due to a lack of clinical specimens. Regarding JCPyV, one amplified strain was characterized as 1B and two NCCRs were defined as “rearranged.” BKPyV VP1 genotyping was performed for seven amplicons, Ia (4/7) and Ib-1 (3/7); the BKPyV NCCR was similar but not identical to the BKPyV Dunlop strain in two cases and to the TU strain in two other cases. The MCPyV NCCR was amplified and sequenced in five CSF samples, and it was characterized as an IIc strain, as seen in HPyVs can be present in different body compartments and/or body fluids. Understanding their tissue tropism may provide insights into the possible pathogenic roles of these viruses in human diseases. In this retrospective study, we investigated the presence of the genome of some HPyVs  in the CSF of patients with neurological diseases, mostly not related to other microbial agents.None of the samples analyzed were positive for HPyV7 and HPyV9. HPyV7 has been previously detected in urine and nasopharyngeal swabs of children undergoing liver transplant  as well As expected, JCPyV genome was detected in the CSF collected from the patients affected with PML. We reported frequent amplifications of BKPyV and MCPyV and a single amplification of the HPyV6 DNA sequences. Even though the prevalence of BKPyV and MCPyV was found to be significantly greater than that of JCPyV, the viral loads and the percentage of cells infected were definitely much higher for JCPyV than the other two HPyVs.6 and 107 copies/load. In another patient, a high BKPyV load was found in the CSF, but with a concomitant infection of EBV and HSV-1, which were the likely etiological agents of meningoencephalitis. In this case, BKPyV could probably play a copathogenic role or be latent in the CNS, and then occasionally be found in the CSF. Concerning the coinfections, it should be emphasized that two other cases showed concomitant infections of BKPyV and herpesvirus or enterovirus.As already stated, this is not the first time that the BKPyV genome has been detected in the CSF or brain tissue. Chittick and colleagues reviewed all the cases of BKPyV isolated from the CNS up to 2013 ; subsequOur results are inconsistent with those from previous reports about the presence of MCPyV in the CSF. However, it should be underlined that, so far, only three papers have reported results about the detection of the MCPyV genome in a total of 168 CSF samples ,30,48. MBecause MCPyV and HPyV6 are part of the human skin microbiota, it is nonetheless possible that the MCPyV and HPyV6 sequences originated from the skin of the patient during CSF collection, as occurs with bacterial contamination of blood after venipuncture ,51.Additionally, the presence of the viral genomes in the sera was determined in only one-third of the positive patients and for only MCPyV and BKPyV. In these cases, only BKPyV was found in one serum sample from a patient affected with neuropathy. Besides this single case, in the other patients HPyVs were probably latent in the CNS, and they did not passively cross the blood–brain barrier (BBB). However, since we are considering diseases often characterized by inflammatory alterations of the BBB itself, assessing the presence of HPyVs in the peripheral district of all the enrolled patients would have been of great interest, to understand whether HPyVs cross the BBB or are already present in the CNS.For a few viral strains, we conducted a molecular characterization. The genotyping results for JCPyV and BKPyV were consistent with those of previously published epidemiological studies, which identified genotypes 1 and I, respectively, as the most represented genotypes within the European population. In addition, the presence of a rearranged NCCR JCPyV infecting PML patients has been widely reported in the literature . The fouOur study: (a) confirmed the neurotropism of JCPyV; (b) indicated the occasional presence of BKPyV and the high prevalence of MCPyV in the CSF of patients affected with neurological disorders, mostly not associated with other microbial agents; (c) did not find evidence of the presence of HPyV7 and 9, and limited presence of HPyV6, in the CSF.The strength of the study arises from the fact that, to the best of our knowledge, this is the largest report on HPyVs presence in the CSF. The description of some CNS-infecting viral strains represents an additional benefit of this study, since there are very few data in the literature defining the genotype and/or the rearrangements of HPyVs other than JCPyV in the CSF.However, the study suffers from some limitations: it would have been more informative to report the results for all the 14 known HPyVs, the data obtained from paired peripheral clinical specimens, such as blood or serum, and the characterization of the sequences of all the amplified viral strains.Whether it is worth searching for several HPyVs in the CSF from patients affected with neurological diseases is still an unanswered, but significant question. The results of our study might indicate the utility of testing BKPyV and MCPyV in addition to the microbial agents usually tested in meningoencephalitis and other neurological diseases.


Trophoblasts are important parts of the placenta and exert vital roles in the maternal‐foetal crosstalk, and sufficient trophoblasts migration and invasion is critical for embryo implantation and normal pregnancy. Macrophages, as the major components of decidual microenvironment at maternal‐foetal interface, can interact with trophoblasts to participate in the regulation of normal pregnancy. Previously, our group have demonstrated that trophoblasts could induce macrophages polarization to M2 subtype by secreting interleukin‐6 (IL‐6); however, the understanding of macrophages regulating the migration and invasion of trophoblasts is limited. In the present study, we used the co‐cultured model to further investigate the effects of macrophages on trophoblasts migration and invasion. Our results showed that co‐culture with macrophages promoted epithelial‐to‐mesenchymal transition (EMT) of trophoblasts, thereby enhancing their migrative and invasive abilities. Further experiments revealed that M2 macrophage‐derived G‐CSF was a key factor, which promoted the EMT, migration and invasion of trophoblasts via activating PI3K/Akt/Erk1/2 signalling pathway. Clinically, G‐CSF was highly expressed in placental villous tissues of normal pregnancy patients compared to patients with recurrent spontaneous abortion, and its expression level was significantly correlation with EMT markers. Taken together, these findings indicate the important role of M2 macrophages in regulating trophoblasts EMT, migration and invasion, contributing to a new insight in concerning the crosstalk between macrophages and trophoblasts in the establishment and maintenance of normal pregnancy. The results showed that co‐culture with macrophages promoted EMT programme of trophoblasts, thereby enhancing their migration and migration. Further experiments revealed that M2 macrophage‐derived G‐CSF was a key factor, which promoted the EMT, migration and invasion of trophoblasts via activating PI3K/Akt/Erk1/2 signalling pathway. Clinically, G‐CSF was highly expressed in placental villous tissues of normal pregnancy patients compared to patients with RSA, and its expression level was significantly correlation with EMT markers. Taken together, these findings indicate the important role of M2 macrophages in regulating trophoblasts EMT, migration and invasion, contributing to a new insight in concerning the crosstalk between macrophages and trophoblasts in the establishment and maintenance of normal pregnancy.22.1Women conducted induced abortion for non‐medical reasons were chose for control group, and patients suffered from recurrent spontaneous miscarriage for two or more times were regarded as RSA group. Twenty‐one RSA patients and 28 normal pregnant women from Renmin Hospital of Wuhan University were included in the study. RSA patients with characteristics including endocrine or metabolic diseases , uterine abnormality, abnormal karyotype or infection (according to the leucorrhoea routine examination) were excluded from the study. The placental villous tissues and decidual tissue samples were collected at the time of surgery, fixed in 4% paraformaldehyde for paraffin‐embedding in blocks or frozen and stored in liquid nitrogen. The procedure was performed with the approval of the internal review and ethics boards of Renmin Hospital of Wuhan University, and informed consent was obtained from all included patients. The baseline characteristics of the patients were summarized in Table 2.22. For macrophage generation, THP‐1 cells were treated with 50 ng/ml Phorbol 12‐myristate 13‐acetate  for 24 h. Macrophages and trophoblasts co‐cultivation was conducted using the non‐contact co‐culture transwell system  for 72h, in which macrophages were seeded in 0.4 μm sized pores inserts, and HTR‐8 was seeded in the 6‐well plate (1.5 × 105 cells per well).The human monocyte cell line THP‐1 was cultured and grown in RPMI‐1640 medium  with 10% foetal bovine serum (FBS) , and the trophoblast cell line, HTR‐8/SVneo (HTR‐8), was grown in DMEM‐F12 medium  with 10% FBS, at 37°C in the presence of 5% CO2O and used at a final concentration of 100 ng/mL. LY294002 (PI3K inhibitors) and PD 98059 (Erk1/2 inhibitors) were purchased from Med Chem Express, China. The anti‐human neutralizing G‐CSF antibody was acquired from R&D Systems, USA.Recombinant human G‐CSF  was dissolved in ddH2.3‐ΔΔCt method. The sequences of primers for RT‐PCR were as following: E‐cadherin, Forward: 5′‐ATTTTTCCCTCGACACCCGAT‐3′, Reverse: 5′‐TCCCAGGCGTAGACCAAGA‐3′; Vimentin, Forward: 5′‐AGTCCACTGAGTACCGGAGAC‐3′, Reverse: 5′‐CATTTCACGCATCTGGCGTTC‐3′; G‐CSF, Forward: 5′‐ATAGCGGCCTTTTCCTCTACC‐3′, Reverse: 5′‐GCCATTCCCAGTTCTTCCAT‐3′; GAPDH, Forward: 5′‐CACTGGGCTACACTGAGCAC‐3′, Reverse: 5′‐AGTGGTCGTTGAGGGCAAT‐3′.The total RNA was isolated using the TRIzol Reagent  according to the manufacturer's instructions. After detection of RNA concentration, 0.5 μg of total RNA was reverse‐transcribed into cDNA using the RT reagent kit , and the cDNA was used for subsequent RT‐PCR using the SYBR‐Green PCR Mix (Takara) by a 7500 Real‐Time PCR . The relative mRNA expression level was calculated using the 22.4Protein extraction and Western blot were performed as we previously reported.2.5The cytokine antibody array agented by Wayen Biotechnologies  was used to detect inflammatory cytokines in the supernatant obtained from HTR‐8, macrophages and the co‐culture model, which contains cytokines associated with invasion including MCP‐1, IP‐10, IL‐6, VEGF, G‐CSF, IL‐15, IL‐1β, IL‐10 and IL‐12. Results were expressed as pictograms per millilitre.2.6The wound healing assay was used to evaluate the migration ability of trophoblasts. A wound was made by dragging the plastic pipette tip across the cell surface when cells were grown to 80% confluence in 24‐well plates, and migrating cells at the wound front were photographed after 24h and 48h. The area of the wound was measured with Image J software .2.7Cell invasion assays were performed using 24‐well transwells  and coated with Matrigel . In total, cells suspended in 500 µl DMEM/F‐12 were added to the upper chamber, while 500 µl DMEM/F‐12 containing 5% FBS was placed in the lower chamber. After 48h of incubation, Matrigel and the cells remaining in the upper chamber were removed using cotton swabs. Cells on the lower surface of the membrane were stained with 0.5% crystal violet for 15 min. Cells from five fields at a magnification of × 200 were counted. All experiments were performed in triplicate.2.8For IHC, serial sections from the human placental villous tissues and the decidual tissues were obtained. IHC was conducted as previously described.2.9U test. Pearson correlation was used to analysis the correlations between the variables in the decidual and placental villous tissues. P values < 0.05 were considered statistically significant.All statistical analyses were performed using SPSS 22. 0  and GraphPad Prism 6.0  for Windows. Groups of discrete variables were compared by means of the Kruskal‐Wallis non‐parametric analysis of variance or Mann‐Whitney 33.1To determine the effect of macrophages on trophoblasts, we utilized an in vitro co‐culture model using the trophoblast cell line HTR‐8 and PMA‐treated THP‐1 macrophages (M0‐Mφ), which were impermeable for cells and allowed the exchange of soluble factors between two cells Figure . HTR‐8 t3.2Previous studies have indicated that macrophages exerted their ability on invasion and migration through the secretion of paracrine factors,Furthermore, a G‐CSF neutralizing antibody was used to confirm the regulatory effect of G‐CSF in HTR‐8. After adding the G‐CSF neutralizing antibody into the co‐culture model, the expression of Vimentin was decreased, while E‐cadherin was increased when compared with G‐CSF treatment group Figure . Consist3.3To determine which downstream signals in HTR‐8 responded to G‐CSF secretion by M2 macrophages, we focused on PI3K, Erk1/2, Akt and P‐38 pathway, which have been reported to be activated in trophoblasts treated with G‐CSF.3.4+ macrophages. Further analysis indicated that the proportion of G‐CSF+CD163+ macrophages in CD163+ macrophages was 52.24%±8.76%, and the proportion of G‐CSF+CD163+ and G‐CSF+CD163‐ in the decidua were 6.78%±3.56% and 15.26%±6.82%, respectively. In addition, a positive correlation was found between the proportion of CD163+ and G‐CSF+ cells (r = 0.465, P = .013; Figure + cells were mainly located on cytotrophoblasts, a few on syncytiotrophoblasts (Figure P = .002; Figure P = .017; Figure Furthermore, we evaluated the expression of G‐CSF in the placental villous and decidual tissues from normal pregnancy. The results found that G‐CSF was expressed on decidual stromal cells, with membrane patterns Figure . To confs Figure . Furthers Figure . PearsonWe summarized our findings in a schematic Figure . Our stu4Successful pregnancy is a complex process involving multiple cells and factors.Macrophages account for approximately 20%‐30% decidual leucocytes, having marked influence on the local environment and thus EVTs function.et al first reported that placental villous and decidual tissues were sources of G‐CSF in 1992,et al that G‐CSF was secreted by decidual cells and decidual macrophages were sources of G‐CSF in 1998.+ cells, which was in accordance with the result that high levels of G‐CSF were associated with increased CD163+ macrophages in triple‐negative breast cancer.+CD163‐ cells were found in the decidua, indicating that in addition to macrophages, other cells in the decidua, such as NK cells, were also sources of G‐CSF.Given the important roles of cytokines in cell‐cell interactions, we screened the changes of a panel of cytokines in the co‐cultured model, and G‐CSF was confirmed as the most significantly up‐regulated cytokine. Subsequent functional assays identified that G‐CSF was involved in macrophage‐induced EMT, migration and invasion of trophoblasts. Previous study from other trophoblast cell lines including Swan 71 cell5In summary, our current findings disclose the important role of macrophages in regulating the EMT programme, migration and invasion of trophoblasts, contributing to a new insight concerning the crosstalk between macrophages and trophoblasts in the establishment and maintenance of normal pregnancy.The authors declare that there is no conflict of interest.Jing Yang, Tailang Ying and Duanying Guo designed the research. Jinli Ding, Chaogang Yang and Yi Zhang performed the experiments and analysed the data. Jinli Ding, Jiayu Wang and Sainan Zhang collected the clinical samples and patients’ data. Jinli Ding, Chaogang Yang and Yi Zhang wrote the draft. Jing Yang, Tailang Ying and Duanying Guo reviewed and revised the manuscript. All authors read and approved the final manuscript.


Parkinson’s disease (PD) is a common neurodegenerative disorder; however, well-established biochemical markers have not yet been identified. This review article covers several candidate cerebrospinal fluid (CSF) biomarkers for PD based on the recent literature and meta-analysis data. The decrease of α-synuclein in PD is supported by meta-analyses with modest reproducibility, and a decrease of amyloid β42 is seen as a prognostic marker for cognitive decline. Tau, phosphorylated tau (p-tau), and neurofilament light chains have been used to discriminate PD from other neurodegenerative disorders. This article also describes more hopeful biochemical markers, such as neurotransmitters, oxidative stress markers, and other candidate biomarkers. Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, and is characterized by resting tremors, bradykinesia, rigidity, and postural instability, as well as cognitive symptoms . PatholoThe current clinical diagnostic criteria of PD are based on existence of parkinsonism and the exclusion of other disorders, and all the tests for PD, such as magnetic resonance imaging or nuclear medicine imaging are positioned as supportive tools . TherefoThe cerebrospinal fluid (CSF) biomarkers for PD have been investigated widely to elucidate the pathophysiology and to support differentiating PD from normal subjects or other neurological disorders, such as multiple system atrophy (MSA)  or progrConfirmed CSF biomarkers have not yet been established; however, some candidate molecules  have been inspected as potential ones. In this article, we aimed to provide a comprehensive overview for the biomarkers for PD and referred to recent meta-analyses for several biomarkers.A previous review divided the biomarker candidates into six categories  . Under eOur review also adopted this categorization; however, the clinical usability was divided into three clinical domains . This categorization is tentative and conceptual, and exceptions are possible . We also annotated the putative functions, roles, or pathophysiological interpretations for each marker in This study is based on a literature search in the database PubMed with the keywords ) on 24 May 2020. Non-English publications were excluded from this review because of the difficulty of assessing the contents as available open resources. Only the most relevant or earliest studies were included in this review and duplicated contents were omitted as well as studies with a small sample size (n < 20).Of the 255 articles identified in the database, 63 were included in this study . The seaWe also focused on the following markers in particular: (1) α-synuclein and its related molecules; (2) dopamine, neurotransmitters, and the metabolites; (3) oxidative stress markers; (4) amyloid beta as a predictor for cognition; and (5) tau, phosphorylated tau, and neurofilament light chains as discriminators for atypical Parkinsonism.The results are summarized in The depletion of dopaminergic neurons in the substantia nigra is an essential pathology in PD, and noradrenergic neurons in locus ceruleus were also observed. Dopamine and noradrenaline decreases are also potential candidates of PD, as well as the metabolites, including dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA). However, the levels of these compounds actually have different sources and meanings . CSF homAs dopaminergic neurons are lost, pathway traffic to the remaining terminals likely increases compensatorily, thereby augmenting dopamine delivery from those terminals to the extracellular fluid; thus, CSF dopamine may underestimate the extent of the loss of neuronal dopamine stores. CSF dopamine concentrations are infinitesimal—sometimes below the detection limit of the assay method . Meta-anOther marker changes , 3-hydroxykynurenine, orexin, and 5-hydroxyindole acetic acid (5-HIAA)) were also reported ,9,45,54.Numerous markers have been investigated to evaluate oxidative stress in PD ,23,24,55·OH), lipid peroxidation, nitrites, nitrates, silicic acid, xanthine, 3-nitrotyrosine products, and 8-hydroxyguanosine (8-OHG)) have also been investigated [Other markers (advanced oxidized protein products (self-oxidized), ceruloplasmin ferroxidase activity, oxidized Q10, Cu/Zn-superoxide dismutase, DJ-1, glutathione S-transferase Pi, glutathione (oxidized), hydroxy radical  was significantly different (decreased) between the control and both neurodegenerative groups (PD and Alzheimer’s disease) but not between neurodegenerative groups . CSF pro2–103 times higher than those found in the CSF [α-synuclein is considered one of the most important targets in this field, and many studies have investigated CSF α-synuclein. The reported control/normal CSF α-synuclein values varied widely; however, meta-analyses showed that the CSF levels of α-synuclein decreased in patients with PD ,32,33,34 the CSF . Synucle123I-ioflupane decreased in the PD group, but this was not correlated with the CSF α-synuclein level [A recent study demonstrated that CSF α-synuclein decreased early in the disease, preceding motor PD. CSF α-synuclein does not correlate with the progression and therefore does not reflect ongoing dopaminergic neurodegeneration. Decreased CSF α-synuclein may be an indirect index of changes in the balance between α-synuclein secretion, solubility, or aggregation in the brain, reflecting its overall turnover . This coin level . Anotherin level .Technical caution and pitfalls for the measurement of α-synuclein should be considered for appropriate use. Perianalytical considerations for biomarker studies in PD have been summarized . α-synucWhy α-synuclein decreases in the CSF of PD remains unknown. Low production, aggregation in the brain, and/or increased clearance of α-synuclein would be possible.Cognitive decline in PD is well known, and several studies reported a relationship between amyloid-beta and cognition. Another recent topic of research interest is the relationship between PD, amyloid β42 (Aβ42), tau, and p-tau, particularly from the viewpoint of cognitive function. One meta-analysis showed that the CSF Aβ42 level in PD with the cognitive impairment (CI) cohort was lower than that in PD with the normal cognition (NC) cohort. Reduced Aβ42 as well as elevated t-tau and p-tau were observed in the PD with dementia (PDD) cohort compared with the PDNC cohort. Therefore, the meta-analysis concluded that amyloid pathology and tauopathy may participate in the development of PDD, which is similar to Alzheimer’s disease .It is unknown whether α-synuclein and amyloid beta can interact; however, a previous study reported no significant correlation between α-synuclein and amyloid beta . There iNeurofilament light chains (NFL) also have the potential to differentiate PD from MSA or PSP, and this is supported by meta-analyses ,58,59,60Other proteins , clusterin, neurofilament heavy chains, YKL-40 (CHI3L1), soluble neuron-glial antigen 2 (NG2) proteoglycan, ubiquitin, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1), apolipoprotein A1, apolipoprotein A2, apolipoprotein ε, transthyretin, glycan isoforms of transferrin (serum-type/brain-type ratio), neuron-specific enolase, myelin basic protein, heart fatty acid binding protein, glucocerebrosidase activity, and neprilysin activity) have also been investigated ,46,62,63Many other candidate markers have been reported  6,9,18,,52,53,61Recent studies focused on the hypothesis that epigenetic mechanisms contribute to PD development and progression. Epigenetics refers to the regulatory mechanisms of gene expression that are not mediated by the DNA sequence itself, but by chemical or allosteric DNA modifications or by the action of regulatory noncoding RNAs. MicroRNAs (miRNAs) are small noncoding RNAs that serve as posttranscriptional regulators of gene expression. They bind to messenger RNA (mRNA) and promote mRNA degradation and/or decrease the translation. One meta-analysis identified four studies for miRNA of CSF; however, no significant signals were found in the CSF, although the study identified 13 significantly differentially expressed miRNAs in the brain (n = 3) and blood (n = 10) . The mosCumulative data, including meta-analyses, support that a decrease of CSF α-synuclein is observed in PD and that a decrease of amyloid β42 is a predictor of cognitive decline in PD. Tau, phosphorylated tau, and neurofilament light chains were able to discriminate PD from other neurodegenerative disorders. The elevation of cytokines  in PD suggested pro-inflammatory mechanisms in the pathogenesis of PD. CSF DOPAC appeared to provide a sensitive means to identify PD. This review could not identify reliable CSF oxidative stress markers in the literature thus far.Further studies are necessary to reveal α-synuclein abnormalities, which may include the transcription, translation, epigenetic regulation, and kinetics . The standardization of measurement techniques is also required to use the practical application of these markers.This review has some limitations. First, the abovementioned meta-analyses showed statistical significance; however, heterogeneity was present in each meta-analysis. Overlapping of the data between PD and the counterpart is present in any marker; therefore, they should be applied carefully. Second, these CSF biomarker samplings were performed mainly while the patients were alive, and, therefore, the diagnosis at the time of CSF sampling was established with clinical criteria but not as a histopathological diagnosis.It is inevitable to have the limitation of pre-mortem differential diagnosis of AD and dementia with Lewy bodies (DLB). Certain studies adopted post-mortem CSF sampling with neuropathologically diagnosed PD subjects ; howeverIn conclusion, the meta-analyses for cerebrospinal fluid biomarkers of PD showed: (1) a decrease of α-synuclein was a marker of PD, (2) a decrease of amyloid β42 was a marker of cognitive decline in PD, (3) the elevation of tau, phosphorylated tau, and neurofilament light chains differentiated between PD and the related disorders, and (4) the elevation of cytokines  was observed in PD. Many other candidate biomarkers were also scrutinized. These findings will aid in the accurate diagnosis of PD and other neurodegenerative disorders and facilitate understanding of the pathogeneses of these conditions. Further studies will be needed to obtain more precise measurements of the biochemical biomarker levels. Any single biomarker cannot lead to a ‘snap-shot’ diagnosis. Combinations of several biomarkers would help with more accurate diagnoses and evaluation of the pathophysiology.CSF biomarkers would aid in understanding the pathophysiological mechanisms, improve the diagnostic accuracy, and facilitate the future development of novel therapies, including disease-modifying drugs.Finally, we refer to the limitation of this article. This work is a narrative review but not a systematic review. Therefore, we admit that this article could contain some selection bias although each report was fairly scrutinized as much as possible. The aim of this work is to provide the latest comprehensive review regarding cerebrospinal fluid biomarkers of PD, to grade the reliance of each marker according to meta-analyses, and to focus on each marker or marker category with respect to future research directions. We agree that the systematic review method is a more reliable scientific approach. However, referring to all reports offsets the conciseness of this article, and even dopamine and the metabolites, which are generally regarded as central issues in the pathophysiology of PD, have no meta-analysis yet. Hence, we did not adopt the systematic review approach in this article.


WHO endorsed the use of epilation as an alternative treatment to surgery for the management of both minor unoperated TT (UTT) and postoperative TT (PTT). However, some trachoma control programmes hesitated to implement epilation citing concerns that it would hamper TT surgical acceptance and result in larger numbers of and stiffer trichiatic eyelashes than the original TT lashes. We investigated the burden and phenotypes of post-epilation trichiatic eyelashes, and willingness to accept surgical management separately in unoperated and postoperative TT cases.rd of the upper eyelid) UTT (170) and PTT (169) from community-based screenings in Amhara Region, Ethiopia. Participants eyes were examined and data on present and future willingness to accept surgical management collected at baseline and every month for 6-months. Eyelashes touching the eye were counted and their phenotypes documented. Participants were trained on how to epilate. Epilation was done by the participants at home and by the examiner during follow-ups when requested by the participant. Follow-up rates were ≥97%. There was evidence of a significant reduction in the burden of trichiatic eyelashes in unoperated , and postoperative  cases 6-month after frequent epilation. Post-epilation trichiatic eyelashes at 6-months had higher odds of being thin , weak , and half-length  than the pre-epilation trichiatic eyelashes in unoperated cases. There was a significant increase in the proportion of weak trichiatic eyelashes  in postoperative cases. In all 6 follow-up time points, 120/164 (73.2%) of unoperated and 134/163 (82.2%) of postoperative cases indicated that they would accept surgery if their trichiasis progressed.We recruited cases with minor  unoperated (170) and postoperative TT (169) cases. We found, in the contrary to these concerns, the post-epilation eyelashes touching the eye were less damaging being fewer in number, thinner, weaker and shorter than the pre-epilation eyelashes. In addition, the majority of both unoperated and postoperative cases indicated that they are willing to accept surgery if their trichiasis progressed.Trachomatous Trichiasis (TT), the blinding stage of trachoma, ranges from few peripheral eyelashes touching the eye to all eyelashes scratching the cornea. TT is mainly treated with corrective eyelid surgery. However, not all TT cases require surgical correction, and some, particularly, those with few eyelashes decline surgery. Epilation, the repeated removal of eyelashes, is a very common clinical and traditional practice in many trachoma endemic settings. The World Health Organisation recommends that epilation can be offered as an alternative management strategy to surgery for patients with few eyelashes touching the eye or refusing surgery. However, some trachoma control programmes hesitated to implement epilation with the concern that it would hamper surgical acceptance and results in larger numbers of and stiffer eyelashes touching the eye than the original TT eyelashes. In this study, we explored if these concerns are true in epilating minor (≤5 eyelashes from the upper eyelid touching the eye or evidence of epilation in <1/3 Trachomatous trichiasis (TT), the blinding stage of trachoma, has variable phenotypes ranging from a single aberrant eyelash touching the eye without entropion (the inward rotation of the eyelid margin) to complete entropion with all eyelashes in-turned towards the eye and scratching the cornea. The WHO nd Global Scientific Meeting on TT indicates that repeat surgery leads to more unfavourable outcomes than the initial surgery.: 1.4[0.9] vs 1.9 [1.1]), and 6-month follow-up (mean lash burden [SD]: 0.2 [0.4] vs 1.1 [1.3]). On the other hand, the 4 postoperative cases who wanted to undergo repeat surgery tended to have higher burden of trichiatic eyelashes than those who didn’t want both at baseline (mean lash burden [SD]: 2.5 [1.9] vs 1.7 [1.2]), and 6 month (mean lash burden [SD]: 1.0 [0] vs 0.6 [0.9]). There were no differences in other trichiasis phonotypes between those who wanted to have surgery and those who didn’t.The majority of the unoperated (120/164 [73.2%]) and postoperative  were aware and 57/75 (44.2%) were unaware that they had trichiasis (p = 0.97). In addition, there was no difference in willingness to accept surgical management at baseline between prior-epilators and non-prior-epilators in both the unoperated  and postoperative  TT groups.CO and vision change results between baseline and follow-ups are presented in During the six-month period, 2,391 trichiatic eyelashes were removed by the examiner: 1,291 (54.0%) in the unoperated group and 1,100 (46.0%) in the postoperative group. Among these, 2,186 (91.4%) were completely epilated and 205 (8.6%) were incompletely epilated. More eyelashes in the postoperative (119 [58.0%]) than the unoperated group (86 [41.9%) were incompletely epilated . Mild pain was felt by 18/168 (10.7%) unoperated, and 3/169 (1.78%) postoperative cases (p = 0.001) during examiner epilation. Among those epilating at home using traditional forceps, 7/20 (35.0%) unoperated and 7/29 (24.1%) postoperative TT cases (p = 0.41) reported pain (any severity).After adjusting for the number of eyelashes touching the eye, the overall number of cases who epilated at home during the six follow-up timepoints was significantly lower than the number epilating at home at baseline in both the unoperated group  and postoperative group   and the number of people managed for TT globally has been reported. Among mard of the upper eyelid) is less likely to be painful and visually troubling than major trichiasis (>5 eyelashes touching the eye or evidence of epilation of ≥1/3rd of the upper eyelid). More than half of the cases had only a single lash touching the eye, which are likely to be missed during screening and impact and surveillance surveys. These data suggest that, firstly, there are still a significant proportion of people with minor TT who are unknown to the health system, a situation likely to persist well beyond 2020. Secondly, surgery is probably a less appealing option for such cases, who do not have both awareness of the trichiasis and a ‘felt need’ for intervention. This is possibly why half of the unoperated and postoperative TT cases in this study considered the less invasive epilation to be an adequate treatment alternative for their trichiasis. Other studies also reported that not having symptom from the trichiasis and symptoms being not bad enough are among barriers for surgical uptake.[Despite a strong trachoma control programme with robust community-based screening and surgical services, nearly half of the unoperated and about a third of the postoperative TT cases enrolled into this study were unaware that they had trichiasis. Minor trichiasis  of a new eyelash would take about 1–2 months , while iDirect comparison of photographs indicated that there is no change in CO grading between baseline and at the 6-month follow-up in both the unoperated and postoperative TT groups, while the field grading in the unoperated group showed mostly a peripheral progression . This is in line with the finding that post-epilation trichiatic eyelashes were more likely to be shorter than the pre-epilation trichiatic eyelashes, and hence be less likely to grow sufficiently long to reach the centre of the cornea. This is similar to the result of a clinical trial we conducted a few years ago in which photographic CO grading showed no change in minor unoperated cases recruited in the same study setting and randomised to the epilation arm while the field grading indicated a moderate variation. We have Epilation significantly improved VRQoL regardless of vision change for both unoperated and postoperative TT cases. The largest improvements were seen in the visual symptom subscale in both groups indicating that epilation is capable of relieving pain and discomfort from the trichiasis. Although, the effect was smaller, the VRQoL finding in this study is similar to what has been reported for TT surgery in earlier studies.,29,30 ThThis is the first longitudinal study with detailed phenotyping of post-epilation trichiatic eyelashes and measuring the effect of epilation in managing postoperative TT cases and its impact on quality of life. We employed logistically intensive frequent follow-ups with high follow-up rates to capture fast growing post-epilation trichiatic eyelashes. The interviews and examinations were conducted by the same persons at each visit, increasing the reliability of the data presented. However, the study has a number of limitations. The frequent follow-up and case management employed in this study may not reflect the realities in programmatic settings. There are no comparison participants that could have helped to explain what would have happened to people without trichiasis, and to people with trichiasis but who refused both epilation and surgery to support the trichiatic eyelash burden and phenotype, corneal opacity, vision, and quality of life change results. Post-epilation eyelash phenotype was studied only for the eyelashes touching the eye. It is possible that the characteristics of some post-epilation eyelashes pointing towards the eye but yet to touch it would be different. However, our purpose was to study post-epilation “trichiatic eyelashes”, and an eyelash not touching the eye is not a trichiatic eyelash. Importantly eyelashes not touching the eye were not epilated at any follow-up time point, therefore would not be captured in any of the next follow-up when they touch the eye given that not many patients were frequently epilating at home. The gradings have been conducted by a single examiner and quality assurance performed within the study team. Although we used a clear grading system eyelash strength and thickness are not always independent of each other. We recognise photo grading on the eyelashes phenotypes by an independent expert would have strengthened the results. The participants were advised to epilate at home after a training on how to epilate but only some practiced home epilation during the follow-up period. However, they were not provided epilation forceps as this was not permitted by the Ministry of Health of Ethiopia. Thus, most cases might have preferred to wait until the next follow-up for their eyes to be epilated by the study team with machine made forceps than by a community member with traditional forceps. This is evident from the fact that the proportion of cases epilating at home have significantly reduced during the follow-up period compared to the baseline. The small frequencies of homebased-epilation could not allow detailed analysis and limited the ability of the study in measuring its impact versus health facility-based epilation. However, in a previous trial that provided training to a friend or relative to do the epilation and two pairs of epilation forceps, we found results comparable with those in the present study mainly on trichiatic eyelash burden reduction and CO changes.,14 SurgiOverall, we found evidence that frequent epilation neither increases the burden of trichiasis nor results in more damaging stiff and thick post-epilation trichiatic eyelashes. In this study setting, cases with minor TT reported a willingness to accept corrective surgery if their trichiasis progressed, potentially contradicting the assumption that the programmatic implementation of epilation would affect surgical uptake. WHO in its Second Global Scientific Meeting on TT, recommended that “It is ethically important to inform the patient with minor trichiasis about both surgery and epilation, and to discuss the benefits and risks of each procedure so that an informed decision can be made”. PatientsS1 Table(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.


Antioxidant/oxidant imbalance has been reported to be related to diminished ovarian reserve (DOR). Vitamin A (retinol), a kind of antioxidant, plays a role in restoring ovarian oxidative damage, while C-reactive protein (CRP) is the classical marker of oxidative stress and has recently been identified as an independent variable that is associated with low anti-Mullerian hormone (AMH) levels in young women with DOR. Additionally, retinol binding protein 4 (RBP4) can be considered a substitute for retinol in healthy, nonobese women. The study aim was to determine the relationship between serum RBP4, high sensitivity C-reactive protein (hs-CRP) concentrations and ovarian reserve in nonobese DOR patients.This study included 24 DOR women and 48 normal ovarian reserve (NOR) women from the reproductive medical center of Renmin Hospital of Wuhan University. The serum RBP4 and high-sensitivity CRP (hs-CRP) levels were measured with ELISA kits.p = 0.025) were decreased, and hs-CRP levels  were increased in the DOR group. Serum RBP4 was positively related to AMH , while hs-CRP was negatively correlated with AMH . after adjustments were made for the covariables, multiple line regression analysis showed that positive association between RBP4 and AMH still existed .RBP4 levels (20,648.36 ± 5475.16 ng/ml vs 23,986.48 ± 5995.64 ng/ml, Decreased serum RBP4 levels and increased serum hs-CRP were observed in DOR patients in our study, and the strong correlation between RBP4 and AMH supports the notion that oxidative stress plays a role in DOR, and that appropriate levels of antioxidant vitamin A may be protective against ovarian reserve dysfunction. Diminished ovarian reserve (DOR) often results in reduced fecundability, accompanied by reduced oocyte quantity and decreased oocytes quality. These phenomena also occur during normal ovarian aging , 2. Due Vitamin A, also known as retinol, is an antioxidant. Its important role in repairing oxidative damage to the ovary in conjunction with other antioxidative vitamins has been extensively studied –9. HowevOxidative stress occurs when the antioxidation system is weakened, at which time, ROS levels increase to induce chronic inflammation and increased levels of inflammatory cytokines. Among them, C-reactive protein (CRP) has been reported to play a key role in this association . Some stWe conducted a cross-sectional study of infertile women between May 2019 and October 2019 in the Reproductive Medical Center of Renmin Hospital of Wuhan University with a total of 72 infertile women aged 21–40 years who met our eligibility criteria. The Clinical Research Ethics Committee of Renmin Hospital of Wuhan University provided ethical approval for study (WDRY2019-K007), and all patients provided informed consent.2); (3) use of steroidal drugs or multivitamins containing vitamin A; (4) unilateral or bilateral ovarian surgery; (5) other factors related to fertility, such as smoking, alcohol consumption and diet; (6) family history of cardiovascular diseases or other metabolic diseases.Before to our hospital, some patients usually have some indicators been tested in other hospitals, we divided all subjects according to their serum AMH values because all infertile women who initially visit our reproductive medical center must undergo AMH testing in our hospital. According to Cohen J, et al. , women wAltogether, 112 infertile women were initially enrolled  kit . The minimum detection limits and sensitivity values were 15.6 ng/mL and 3.9 ng/ml, respectively, for RBP4, and 0.625 ng/ml and 0.156 ng/ml, respectively, for hs-CRP. The intra-assay variation coefficients of RBP4 and hs-CRP are both less than 8%, and inter-assay variation coefficients of RBP4 and hs-CRP are both less than 10%.P < 0.05. All data were processed and analyzed using SPSS 25.0 software.According to the normal distribution of variables, continuous variables are presented as means ± SDs or medians ± interquartile ranges (IQRs), and categorical variables are expressed as numbers (percentages). Continuous variables were analyzed by t-test or the Mann Whitney U-test, and categorical variables were assessed by the chi-square test. Univariate and multivariate linear regression analyses were then performed, while controlling for age and BMI, to further evaluate the relationship between RBP4, and hs-CRP concentrations and AMH levels. The statistically significant setting was P < 0.001). As expected, the DOR group had a higher day-3 FSH level , lower AMH level , and lower total antral follicle count  than the NOR group. After IVF treatment, fewer oocytes were retrieved from the DOR group than from the NOR group . In addition, compared with that of the NOR group, the RBP4 levels  were decreased, and the hs-CRP levels  were increased.Demographic, and related variables of IVF treatment and laboratory parameters for all participants are listed in Table r = 0.518, p = 0.000) and between hs-CRP and AMH  ; however, a positive relationship between RBP4 and AMH still existed   Fig. . After aIn this cross-sectional study, the concentrations of RBP4 and hs-CRP in the plasma of DOR patients were explored for the first time. We found lower RBP4 levels and higher hs-CRP levels in DOR subjects and a positive association between RBP4 and AMH. After adjustments were made for age and BMI as covariates, this relationship still existed. hs-CRP was not associated with AMH; therefore, RBP4 seems to be associated with ovarian reserve.Like vitamin E, vitamin A (retinol), is an antioxidant . Because2 or less in our study. Additionally, those at risk of any metabolic diseases were excluded. Therefore, to a certain extent, we controlled for the factors that led to changes in apo-RBP4 levels so that RBP4 levels could better predict blood retinol levels. This view was verified in another study. Hermsdorff et al. [2).Due to limitations of commercial Elisa kits, it is impossible to distinguish the above two kinds of RBP4 and evaluated the total RBP4 levels. However, in vitro studies have revealed that apo-RBP4 induced inflammatory cascades . Clinicaf et al.  found thOvarian dysfunction caused by oxidative stress has been reported to be associated with DOR. Long-term low-dose use of antioxidants containing vitamin A can protect the ovary from ROS induced by oxidative stress , 6, 9. A2 or less, this criterion was more stringent than the standards used in previous studies. Our study included only women with normal serum levels of lipids and fasting glucose and women with no family history of cardiovascular diseases because abnormalities in these biochemical parameters and a family history of cardiovascular diseases may impact the plasma levels of CRP and AMH.Oxidative stress induces an abnormal immune response, together with an increase in inflammatory markers. Among them, CRP is a classic marker of chronic inflammation  and has To the best of our knowledge, this study is the first to explore the potential connection between the ovarian reserve marker AMH and RBP4, hs-CRP. This research has the following advantages: First, we assessed a group of patients within a normal BMI range and who were under the age of 40 years and had no metabolic risk factors, to eliminate the additional effects of these factors on AMH and biochemical indicators in this experiment. Second, all of our blood samples were taken before IVF started, considering that different doses and kinds of ovulation drugs may have unknown impacts on inflammation and oxidative stress, thus minimizing the potential measurement bias caused by IVF treatment and providing the possibility for an accurate assessment of ovarian function.Our research has limitations. First, the cross-sectional character of the study that did not able to follow-up of participants at a later stage. Second, we did not perform a direct analysis of retinol but did use RBP4 instead. This was because RBP4 is more stable than retinol and because RBP4 detection kits are less expensive and more easily obtained than retinol detection kits. Third, we did not go on to assess this association in the follicular fluid of the patients due to the small sample sizes of the follicular fluid we collected. Currently, we are still enrolling patients who meet the criteria and collecting both blood and follicular fluid for further research. Fourth, the small number and scope of the research population is mainly related to our strict inclusion and exclusion criteria. It is necessary to further expand the research to include a broader patient group. Nevertheless, our results may provide insight into the potential mechanisms that regulate ovarian reserve.In conclusion, decreased serum RBP4 levels and increased serum hs-CRP were first observed in DOR patients in our study, and RBP4 was positively correlated with the ovarian reserve marker AMH. This underlines the fact that oxidative stress plays a role in DOR, and vitamin A may be involved in the recovery of ovarian reserve in DOR patients. However, this association may mainly occur in nonobese women, and whether this phenomenon also occurs in obese DOR patients remains to be determined. Further research should be done in a larger and more diverse population.


Background: Plaque rupture is an important etiology for symptomatic carotid stenosis. The role of calcification in the plaque vulnerability has been controversial. We aimed to detect the geometric features of calcifications in carotid plaque and to examine its association with plaque rupture.Methods: Optical coherence tomography assessment of carotid plaque was performed in 88 patients. Calcification shape was evaluated through quantitative measurements of the long and short axis, area size, circumference, calcification arc, and longitudinal length. Calcification location was analyzed through the distance to the lumen. Furthermore, we developed idealized fluid-structure interaction models to investigate the association of calcification shape and plaque stress.Results: A total of 33 ruptured plaques and 30 non-ruptured plaques were recognized. Ruptured plaques had more multiple calcifications and protruded calcifications. The calcifications in the ruptured plaques displayed a remarkably lower long-axis/short-axis (L/S) ratio than in the non-ruptured plaques (p = 0.001). We classified calcification shape into crescentic calcification (L/S > 2.5) and rotund calcification (L/S ≤ 2.5). Rotund-shaped calcifications were more common in ruptured plaques than in non-ruptured plaques (p = 0.02). Superficial calcifications with minimal distance to the lumen ≤ 50 μm accounted for 79.4% of all calcifications in the ruptured plaques, and only 7.7% in the non-ruptured plaques (p < 0.001). Biomechanical analysis showed that the plaque with rotund-shaped calcification developed 7.91-fold higher von Mises stress than the plaque with crescentic calcification.Conclusions: Superficial calcifications and rotund-shaped calcifications are associated with carotid plaque rupture, suggesting that calcification location and shape may play a key role in plaque vulnerability. Carotid atherosclerosis remains an important cause of stroke and transient ischemic attack. Ruptured plaques, defined as lesions with a structural defect in the fibrous cap with the underlying necrotic core, are often associated with ulcerated plaques on carotid angiography . Disruptin vivo. Several studies have utilized OCT to investigate lipid-rich component, thrombus and fibrous cap thickness in the carotid artery plaques  has lately been developed as a high-resolution imaging modality which provides maximum axial resolution of 15–20 μm, thus offering a unique tool to image the detailed structure of arterial plaque  plaques –12. But n = 13), poor image quality (n = 12) and non-atherosclerotic etiologies (n = 2). Finally, 63 lesions in 62 patients were enrolled in the final analysis.From April 2013 to November 2018, 88 consecutive patients with 90 cervical internal carotid artery (ICA) stenosis who evaluated by OCT at Department of Neurology, Jinling Hospital were enrolled. The application of OCT to human cerebral angiography was approved by the Ethics Committee of Jinling Hospital. Informed consent was obtained from all patients before digital subtraction angiography (DSA) and OCT imaging. Twenty-seven lesions in 26 patients were excluded for the following reasons: incomplete target lesions imaging  lesion were defined as a documented history of transient ischemic attack, amaurosis fugax, or stroke in the vascular territory supplied by the ICA within 6 months . TIA wasA DynaCT angiography scanner  was used for DSA examination. The grade of carotid stenosis was evaluated by using angiography according to North America Symptomatic Carotid Endarterectomy trial (NASCET) criteria . FrequenAll images were analyzed by 2 independent investigators (XS and RL) who were blinded to the clinical presentations and angiographic findings. To determine the reproducibility of OCT qualitative assessments, data were analyzed by 2 independent analysts (XS and RL) and repeated 1 month after initial analysis by the same analyst (XS). Images were considered non-analyzable if there was a serious artifact, or if intraluminal blood impaired the assessment of a continuous 270° arc .Plaque rupture was identified by the presence of fibrous cap discontinuity with a clear cavity formed inside the plaque . RuptureThe minimum lumen area (MLA) and minimum lumen diameter (MLD) of each segment was measured, as well as reference vessel area (RVA) and reference vessel diameter (RVD). References were defined as the most “normal-appearing” segment distal to the lesions on the basis of OCT-derived NASCET criteria , 16. PerCalcification was recognized as well-delineated, low signal attenuation with sharp borders . We measThe minimal depth between the inner edge of calcification and the luminal surface was measured 3 times, and the average value was calculated. Length of calcification was calculated by the total slice number multiplied by the frame interval. The angles of each calcification were analyzed every 1 mm using the center of the mass of the lumen. Maximum calcification arc was defined as the widest angle in which calcifications were detectable, and mean calcification arc was defined as total calcification angle divide for the number of frames presenting with calcification . CalcifiWe constructed the two idealized models with our defined types of calcification morphology by three-dimensional, two-way fluid-structure interaction (FSI) computational model in Unigraphics NX 12.0  and the finite element program ANSYS Workbench 19.2 . We assessed stress on a plaque that comprises three main tissue types: calcification, fibrous and a blunt cresent-shaped lipid core. In this model, the lumen and vessel diameters were chosen to be 3 and 10 mm, respectively, and the vessel wall thickness was 1.5 mm. The lumen was modeled with an eccentricity of 2 mm with respect to arterial center. The lipid core in the maximum cross-section slice was constructed by extending a 140° crescent with thickness of 2.3 mm. The idealized models only differed in calcification morphology. The minimum depth between the inner edge of calcification and lumen was 0.1 mm.E) in circumferential (θ) and radial (r) directions, νrθ and νrzthat are the Poisson ratios in r-θ and θ-z planes, respectively, as well as Grθ that is the shear modulus in r-θ plane. The material constitution and corresponding parameters of all tissues were adapted from previous studies . Receiver-operating characteristic curve (ROC) analysis was performed to assess the best cut-off value of L/S and depth of each calcification to predict plaque rupture using MedCalc Version 18.2.1 .Continuous variables with normal distribution were described as mean ± SD, and variables with skewed distribution were described as median (the 25th to the 75th percentile). The distribution of continuous variables was tested by the Kolgormonov-Smirnov test. Comparisons for continuous variables were performed using an independent samples p = 0.03). There were no significant differences in age, clinical and biochemical parameters between patients with or without plaque rupture . Nevertheless, the area stenosis of lesions with ruptured plaques were similar with lesions with non-ruptured plaques (p = 0.26).A total of 33 ruptured plaques and 30 non-ruptured plaques were recognized from 62 patients . Rupturep < 0.001). 75.8% of the ruptured plaques presented with TCFA, as a presence of fibrous cap <65 μm illustrated by OCT. By contrast, only 23.3% of the non-ruptured plaques contained TCFA. Lipid-rich plaque was more common in the ruptured plaques than in non-ruptured plaques (81.8 vs. 40.0% p = 0.001). The incidence of thrombus  and neovascularization  were likewise significantly higher in ruptured plaques. Macrophages and cholesterol crystals appeared similarly in plaques with or without rupture.The minimal fibrous cap thickness was markedly smaller in ruptured plaques than in non-ruptured plaques (p = 0.04). However, the incidence of plaque disruption, thrombus, macrophage accumulations and cholesterol crystals appeared similarly in lesions with mild or severe stenosis (We further classified plaques into mild stenosis lesions (<50%) and severe stenosis lesions (≥50%). Lesions with severe stenosis were more presented with lipid-rich plaque, overlying thinner fibrous cap, than lesions with mild stenosis . However, multiple calcifications were more common in ruptured plaques than in non-ruptured plaques . 47.1% of the calcifications in the ruptured plaques were rotund-shaped calcifications. Meanwhile only 7.7% of the calcifications in the non-ruptured plaques were rotund-shaped calcifications (p = 0.02).We measured the long axis (L) and short axis (S) of the semilunar-shaped calcifications in the axial OCT images . The calp < 0.001). The potential cut-off values of minimal distance predicting plaque rupture were also measured . Superficial calcifications accounted for 79.4% of all calcifications in the ruptured plaques, and only 7.7% in the non-ruptured plaques (p < 0.001). The distribution of calcification shape and location in the ruptured and non-ruptured plaques are shown in The depth from the calcification to the lumen was also measured by OCT. Remarkably, the distance to the lumen of non-ruptured plaques was more than four times the length of the distance in ruptured plaques  and surface circumference/area ratio . Such difference may be relevant to the calcification shape. Geometrically, the calcification with smaller L/S means rounder calcification with less surface circumference/area. Moreover, lesions with ≥50% stenosis also showed lower calcification area, circumference and calcification index, as well as lower surface circumference/area (p = 0.04). The longitudinal calcification length, calcification arc and calcification index were similar between ruptured and non-ruptured plaques. No difference was observed in the number of spotty calcifications between two groups. Nevertheless, ruptured plaques had more protruded calcification than non-ruptured plaques .Other geometric features of calcifications were also gauged. Calcifications in the ruptured and non-ruptured plaques had similar area size. However, calcifications in ruptured plaques had smaller circumference  The frequency of calcification was similar between ruptured and non-ruptured plaques. Ruptured plaques had more multiple calcifications and protruded calcifications. (2) Superficial calcifications and rotund-shaped calcifications were independently associated with plaque rupture. (3) Rotund-shaped calcification exerted significantly higher von Mises stress on the plaque in the idealized FSI models.Most previous studies differentiated between calcified and non-calcified plaques, spotty and large calcifications, single and multiple calcifications to determine the role of calcification in the plaque stability. With OCT, we detected a more detailed architecture of calcifications in the carotid plaque, including calcification shape, location, quantitative measurements of semilunar axis, area size, circumference, cross-sectional arc, longitudinal length, and etc. Since calcifications on cross-sectional planes are somewhat moon-shaped at different phases, we first classified calcifications into crescentic or rotund calcifications based on their L/S ratios. Rotund-shaped calcifications strongly associated with plaque rupture. Biomechanical analysis using idealized FSI models may provide an explanation for this association. Rotund-shaped calcifications exerted more stress on the calcification-fibrous boundary and the luminal surface.Geometrically, the calcification with smaller L/S means rounder calcification with less circumference/area. Thus, a rotund calcification will have less fibrous interface than a crescentic calcification with same calcified contents (same area). Calcified plaque is at least 4 to 5 times more stiff than cellular plaque . Failuresuperficial calcifications. Superficial calcification was first introduced in coronary artery by means of intravascular ultrasound as calcification located at the intimal-lumen interface or closer to the lumen than to the adventitia  were evaluated from severe stenosis (>70%), which has been reported to linked with severe calcifications located the outside layer of the atheroma . The lesSuperficial calcifications with rotund shape in carotid atherosclerotic plaques are associated with plaque rupture, suggesting that calcification shape and location may play a key role in plaque vulnerability.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Jinling Hospital. The patients/participants provided their written informed consent to participate in this study.XS: study design, OCT image collection and analysis, statistical analysis, and manuscript drafting. YH and RL: OCT image collection and analysis. ML: image collection and interpretation of results. QY, FW, and XX: data collection. YX: critical revision of manuscript. RY: study design, interpretation of results, and critical revision of manuscript. XL: study design. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BackgroundEnd-stage renal disease (ESRD) creates a great burden on the quality of life. Patients after kidney transplantation have been reported to have a greater quality of life and better outcomes health outcomes. Therefore, it is important to optimize the best method of following well-constructed criteria such as the expanded criteria donor (ECD) to reduce the chances of rejection rate and deaths post-transplantation particularly in elderly patients in conjunction with the kidney profile donor index (KDPI).MethodsThis is a retrospective descriptive study of all patients who received kidney transplantation from a deceased donor from the ECD as well as ECD with donation after cardiac death (DCD) at St. Joseph Health Care Hospital over a 24 month time period from January 2017 to January 2019. All adult recipients from standard criteria donor (SCD) and living donors were excluded from the study.ResultsThe study included 60 patients with 36 (60%) from the ECD and 24 (40%) were from the ECD/DCD group. The most common cause of ESRD among recipients was diabetes mellitus (DM) involving 23 (38.3%) of the patients. The creatinine outcome was the highest in the ECD/DCD group at one month (211 ± 71) and the lowest creatinine recorded was also in the ECD/DCD at 12 months (160 ± 78). Lastly, only four patients died in 12 months and only six recipients reported graft loss over 12 months.ConclusionDescriptive data of the included ECD/DCD showed increase trend in survivability of the recipients when used among the elderly, giving us more insight on the benefits of ECD/DCD transplantation. Kidney transplantation proves to be the best treatment of choice for most patients with end-stage renal disease (ESRD). Generally, survival and quality of life are found to be superior in allograft recipients compared with patients on the deceased donor waitlist . This beThe Organ Procurement and Transplantation Network (OPTN) instituted a formalized definition of marginal kidneys in 2002 with the advent of ECD . AccordiStudies suggest that kidneys from donors over the age of 60 may have suboptimal graft survival, with a three-year graft survival being only 58%  while a PatientsWe included in this retrospective descriptive study all adult renal transplant recipients who received a deceased donor kidney from an ECD from January 2017 to January 2019 at St. Joseph Health Hamilton, Ontario in Canada. We excluded the living kidney transplant recipients, non-ECD donors, and KDPI that cannot be calculated based on the donor information available. The study protocol was approved by the Hamilton Integrated Research Ethics Board. Data was collected from Ontario’s Trillium Gift of Life Network (TGLN) donor charts and recipients’ charts in the St. Joseph Health Care System using Epic .Study populationA total of 60 adult recipients (age ≥ 18 years) who received an ECD kidney transplant from January 2017 to January 2019 were eligible for the study including those with a prior kidney transplant and ECD/DCD donors. Adult recipients from SCD and living donors were excluded from the study.OutcomesThe primary outcomes were renal function at 12 months measured by serum creatinine, total graft loss defined as the time from transplantation to a composite of return to chronic dialysis, pre-emptive re-transplant or death with graft function, all-causes of mortality, and primary nonfunction graft which was defined as a failed function of the transplanted kidney that necessitated continued maintenance dialysis. The secondary outcomes were incidence of delay graft function (DGF) defined as kidney function that ultimately supported the patient but necessitated post-transplantation dialysis within seven days after kidney transplant, biopsy-proven allograft rejection, the incidence of cytomegalovirus (CMV), and BK virus (BKV) in the first 12 months after the kidney transplant and length of stay in the hospital after the transplant.Out of the 60 patients, 36 (60%) belonged to the ECD while 24 (40%) belonged to the ECD and DCD group. Regarding age, the mean age of the recipient group was 68 ± 7.6 years while the mean age of the donor was 62 ± 4.8 years. In addition, the mean body mass index (BMI) of the recipients and donors was 28 ± 6 years and 29.4 ± 6.8 years, respectively. As well, 23 recipients (38.3%) had diabetes mellitus (DM) as the cause of ESRD making it the most common cause, followed by glomerulonephritis with 15 reported cause (25%), and the least common cause was polycystic kidney disease in three patients (5%). Most recipients had hemodialysis at baseline therapy 43 (71.7%) compared to only 15 (25%) towards peritoneal dialysis and two (3.3%) patients with a failed kidney transplantation. Regarding the choice of an induction agent, 46 (76.6%) patients received basiliximab alone, 11 (18.3%) recipients received anti-thymocyte globulin (ATG), and only 3 (5%) received both (Table Donors were mostly of white ethnicity 51 (85%) with most of them in the ECD and DCD group 23 (95.8%). Meanwhile, 28 individuals from the donor group were hypertensive compared to only six diabetics. Regarding the cause of death in the donor groups, cerebrovascular accidents were the most dominant with 38 donors (63.3%) followed by head trauma 8 (13.3%), anoxia 6 (10%), other 6 (10%) and central nervous system tumor 2 (3.3%) , nine from the ECD, and 11 from the ECD and DCD group. When comparing CMV and BK viremia, they were almost similar with 19 (32%) individuals infected with CMV and 17 (28.3%) reported BK viremia. However, CMV viremia was higher in the ECD group with 11 recorded infections compared to 10 BK viremia cases in the ECD and DCD group. Regarding transplant rejection outcome, only four recipients were reported to have rejection at the 12th month with only one reported antibody-mediated rejection in the ECD, and three had T-cell-mediated rejection  without diabetes. The patients survived four months after his transplant and died due to small bowel obstruction accompanied by sepsis. The second reported case was a 74-year-old male with diabetic neuropathy as the culprit of ESRD. Similarly, he had been on dialysis for five years and passed away two months after the transplant due to small bowel perforation and sepsis Table .As previously stated, four recipients reported a rejection pattern of 12 months without any reports of non-adherence. The first case was an antibody-mediated rejection type with no donor specific antibody. The patient’s calculated panel reactive antibody (cPRA) was 78% and the HLA mismatch was reported as 11/18 0DQ 0DR, and the induction agent of choice was thymoglobulin. The second case, however, was similar except that the rejection pattern was T-cell mediated rejection with cPRA of 30% and an HLA mismatch of 13/18 0DR 1DQ. Also, the last two cases had basiliximab as an induction agent with no reported cPRA 0% and a borderline T-cell mediated rejection (Table Lastly, death censored graft loss was immediate in three cases and was delayed in the fourth one 10 months after the transplant. The first case was a 69-year-old patient with ESRD caused by urinary tract infection and had a delayed rejection that occurred after 10 months. The main cause of the allograft loss was recurrent CMV viremia and the donor was 69 years old from the ECD group with a KDPI of 86%. The second case was a 78-year-old patient with ESRD caused by hypertension with an immediate rejection of the graft. The main cause of the allograft loss was a primary nonfunctional cause where the donor was 67 years old from the ECD and DCD group with a KDPI of 89% and was severely atherosclerotic. The third case was that of a 74-year-old patient with FSGS as the cause of ESRD and immediate rejection. The donor was 66 years old from the ECD group with a KDPI of 95%. Lastly, the final case of a 47-year-old patient with ESRD caused by FSGS who also had an immediate rejection and the donor was 71 years old from the ECD group with a KDPI of 91% (Table The growing gap between demand and supply for kidney transplants leads to renewed interest in the use of ECD kidneys to increase the donor pool. In Canada, kidneys from deceased donors makes up approximately 60% of the donor pool over the last decade . AlthougAccording to Young et al., total graft loss was 10% vs 11.4% in the first-year post transplantations . In compDescriptive data of renal transplantation recipients with ECD kidneys at St. Joseph Hospital resulted in initial favorable outcomes when compared with other programs in Canada as well as global programs. The use of these organs should continue to be more widely considered given the high morbidity and mortality experienced by patients on dialysis.


Classical swine fever (CSF) is a highly contagious viral disease of pigs, including wild boar. It is regarded as one of the major problems in the pig industry as it is still endemic in many regions of the world and has the potential to cause devastating epidemics, particularly in countries free of the disease. Rapid and reliable diagnosis is of utmost importance in the control of CSF. Since clinical presentations of CSF are highly variable and may be confused with other viral diseases in pigs, laboratory diagnosis is indispensable for an unambiguous diagnosis. On an international level, well-established diagnostic tests of CSF such as virus isolation, fluorescent antibody test (FAT), antigen capture antibody enzyme-linked immunosorbent assay (ELISA), reverse-transcription polymerase chain reaction (RT-PCR), virus neutralization test (VNT), and antibody ELISA have been described in detail in the OIE Terrestrial Manual. However, improved CSF diagnostic methods or alternatives based on modern technologies have been developed in recent years. This review thus presents recent advances in the diagnosis of CSF and future perspectives. Classical swine fever (CSF), a list-A disease classified by the World Organization for Animal Health (OIE), is considered as a transboundary animal disease by the Food and Agriculture Organization of the United Nations (FAO) . The disPestivirus in the Flaviviridae family . Different from the CSFV neutralizing monoclonal anti-E2 antibody based cELISA for sero-monitoring of C-strain vaccination at a population level [rns mAb-based cELISA is a valuable tool for measuring and differentiating immune responses to C-strain vaccination and/or infection in pigs. The data about the establishment and validation of this C-strain specific cELISA will be published separately at a later date. In brief, suitable tools for serological DIVA are available. However, there is room for improvement, especially with respect to cross-reactivity issues.Additional approaches or alternatives are undergoing development or further optimization. These include the multiplex microsphere immunoassay , which ised Erns . Recentlon level , this noUser-friendly, cost-effective, rapid, and reliable POC diagnostics  are indispensable tools for immediate decisions of effective and evidence-based disease control strategies . For exaPOC diagnostics showed advantages in rapidity and portability, which are the most important parameters considered by farmers and veterinarians . It is fpestiviruses is critical.Although commercial and in-house diagnostics (antigen detection and antibody detection) of CSF are available, there is still room for improvement. The authors suggest that the following aspects should be considered: (i) Continuously improving the sensitivity, specificity, costs, speed, automation, and POC is necessary; (ii) Reference materials  should be produced and be accessible for validation of the developed CSF diagnostics; (iii) The development of DIVA diagnostics without cross-reaction with antibodies induced by other The other emerging infectious diseases, such as the spreading of African swine fever in Asia and the ongoing pandemic of Coronavirus disease 2019 (COVID-19), may shift focus away from the CSF ,88. Howe


Spontaneous cerebrospinal fluid (CSF) rhinorrhea represents an important clinical entity that is being observed with increasing prevalence, ranging from 14 to 55%. Spontaneous CSF rhinorrhea is associated with elevated intracranial pressure (ICP), which is rarely stopped without surgical intervention. Endoscopic endonasal repair is typically warranted for CSF rhinorrhea. However, the recurrence rate of CSF leaks after the endoscopic endonasal repair of skull base defects due to ICP is usually high. We describe a 25-year-old man without a history of head injury, tumor, or obesity. The onset of his symptoms occurred in 1 week in the form of a persistent clear left nostril rhinorrhea. Computed tomography (CT) and magnetic resonance images (MRI) showed signs of CSF in the left sphenoidal sinus, meningocele in the left frontal sinus, empty sella, hydrocephalus, and Chiari I malformation (CIM). Cine-MRI revealed the flow of CSF was obstructed at the aqueduct and the outlet of the fourth ventricle. Endoscopic third ventriculostomy (ETV) was performed for the patient with obstructive hydrocephalus. Post-operative CSF pressure measurement demonstrated elevated ICP. The patient still had case of CSF rhinorrhea, and subsequently underwent lumboperitoneal shunt (LPS) for treatment of ICP. The patient showed a prompt resolution of CSF leak. Ten months later, the patient showed a significant improvement in terms of his herniated tonsil and cessation of CSF rhinorrhea. Spontaneous cerebrospinal fluid (CSF) rhinorrhea occurs in the absence of traumatic, tumor, or iatrogenic disorder, which usually makes up about 3–4% of CSF leaks . Over thEndoscopic endonasal repair is the preferred surgical intervention for spontaneous CSF leaks, but reported recurrence rates vary widely in the literature, ranging from 25 to 87% . MultiplSo spontaneous CSF rhinorrhea management requires adjunctive measures to supplement the surgical repair of the leak site including conservative treatment, repeated lumbar punctures, and/or CSF diversion to reduce the ICP .Optimal treatment strategies for spontaneous CSF rhinorrhea are not clearly established. In the present case, we describe the case of a patient who presented with spontaneous CSF rhinorrhea due to an elevated ICP. The patient was managed successfully without repair of the dural defect via endoscopic third ventriculostomy (ETV) and lumboperitoneal shunt (LPS).The patient was a 25-year-old man with no history of head injury, tumor, or obesity. He presented with complaints of a watery discharge from his left nostril for 1 week. He reported that the nasal discharge was associated with a headache without fever. On physical examination, no other abnormalities were noted.The head preoperative computed tomography (CT) of the patient showed abnormal density in the left frontal sinus and left sphenoidal sinus. Moreover, the brain magnetic resonance images (MRI) demonstrated bilateral enlarged lateral ventricles because the Evans’ index (EI) is greater than 0.3 (EI = 0.42) . T2-weig2O by lumbar puncture. A lumbar drain was inserted in immediately post operation to last 1 week in order to reduce CSF pressure. No CSF rhinorrhea was observed during the lumbar drain period. However, it reoccurred afterward. Then LPS was performed to decrease ICP and rhinorrhea cessation on day 1 after shunting.An ETV was performed for reconstructing the CSF circulation . Yet hisAn MRI at 10 months after the surgery showed improved cerebellar tonsillar hernia, signs of CSF in the left sphenoidal sinus, and that meningocele had also disappeared . The patSpontaneous CSF rhinorrhea without an identifiable inciting event rarely resolves spontaneously, and if left untreated may increase the risk of ascending bacterial meningitis. The pathologic mechanism of spontaneous CSF leaks is still not well understood. Over the last decade, mounting evidence indicates that elevated ICP could be a contributing factor for spontaneous leaks .Although endoscopic repair methods can achieve a more than 90% success rate for CSF leaks as a whole, the historical success rates for spontaneous leaks were much worse . Many reAt present, interventions to reduce ICP have been more extensively studied in patients with spontaneous CSF leaks. Previous studies reported that patients are generally obese middle-aged females and may present with elevated ICP and symptoms of CSF leaks. However, our patient was a 25-year-old man without a history of trauma or obesity. Recent studies support the role of reducing ICP via medication, weight loss, or CSF diversion to improve the recurrence rates of spontaneous CSF leaks after endoscopic repair . However2 images. T2 images were hyperintense in the left sphenoidal sinus compared to the brain, showing the signaling characteristics of CSF. Patients with spontaneous CSF leaks commonly have radiologic evidence of elevated ICP, including meningocele formation, ranging from 50 to 100% (We reported a case of CIM with progressive hydrocephalus and syringomyelia that was successfully treated by ETV . ETV is  to 100% . The mos to 100% . These d2O was measured by lumbar puncture. Lumbar drains have been used to transiently reduce CSF pressure in patients with high pressure CSF leaks, with increased success rates of CSF fistula repair (Therefore, in the present case, restoring CSF flow should firstly considered by ETV. Treatment strategies for the patient were established . Howevera repair . In the a repair . Even ifa repair . There aa repair . LPS area repair . First aCessation of the CSF rhinorrhea was observed at a 10 months’ follow-up after shunt surgery. LPS were previously associated with the development of CIM, especially in pediatric patients . HoweverThe most important factor for successful treatment of spontaneous CSF rhinorrhea and CIM in this patient was reducing ICP through ETV and LPS. A decrease of CSF pressure alone for the treatment could be considered in some cases, as illustrated by our observation. However, further studies are necessary.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by the Ethical Committee of Nanjing Jinling Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent has been obtained from the patient for the publication of any potentially identifiable images or data included in this article.CM contributed to the conception of the study. CT performed the investigation and data curation. KF modified the grammar mistakes. JZ, JY, ZC, and XC contributed significantly to the analysis and manuscript preparation. LQ helped to perform the analysis with constructive discussions.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.