The skull base region is anatomically complex and poses surgical challenges. Although many textbooks describe this region illustrated well with drawings, scans and photographs, a complete, 3D, electronic, interactive, realistic, fully segmented and labeled, and stereotactic atlas of the skull base has not yet been built. Our goal is to create a 3D electronic atlas of the adult human skull base along with interactive tools for structure manipulation, exploration, and quantification.Multiple in vivo 3/7 T MRI and high-resolution CT scans of the same normal, male head specimen have been acquired. From the scans, by employing dedicated tools and modeling techniques, 3D digital virtual models of the skull, brain, cranial nerves, intra- and extracranial vasculature have earlier been constructed. Integrating these models and developing a browser with dedicated interaction, the skull base atlas has been built.This is the first, to our best knowledge, truly 3D atlas of the adult human skull base that has been created, which includes a fully parcellated and labeled brain, skull, cranial nerves, and intra- and extracranial vasculature.This atlas is a useful aid in understanding and teaching spatial relationships of the skull base anatomy, a helpful tool to generate teaching materials, and a component of any skull base surgical simulator. The base of skull, or skull base, forms the floor of the cranial cavity and separates the brain from other structures of the face and neck. It is composed of five bones: ethmoid, sphenoid, occipital, frontal, and paired temporal. The skull base is subdivided into three distinct regions called fossae: the anterior, middle, and posterior cranial fossae. The base of skull not only separates the intracranial content and the facial compartment from the upper respiratory and digestive tracts, but it also enables the passage of vital neurovascular structures entering and exiting the brain. The skull base region is anatomically complex and poses surgical challenges for neurosurgeons and otolaryngologists because of proximity to important structures, such as the brain, cranial nerves, orbits, and carotid arteries, among others. Moreover, the skull base provides access to hardly accessible intracranial lesions located adjacent to the brainstem . TherefoThe skull base has been comprehensively described in many textbooks –4, Gray’Although many textbooks describe the skull base region well illustrated with drawings, scans, and photographs of anatomical dissections as well as animations and other electronic media have been developed for studying the skull base region, a complete, truly 3D, electronic, interactive, realistic, fully segmented and labeled, decomposable, and stereotactic atlas of the skull base has not been created yet. Therefore, there is a great need for a 3D atlas covering the practical neuroanatomy of the skull base region. The goal of this work is to create a 3D electronic atlas of the adult human skull base along with interactive tools for structure manipulation, exploration navigation, and quantification.A 3D atlas of the adult human skull base region has been created from 3D digital virtual models of the skull, brain, cranial nerves, intracranial vasculature, and extracranial vasculature. These models have been built earlier from in vivo multiple 3 and 7 Tesla (T) magnetic resonance imaging (MRI) and high-resolution computed tomography (CT) scans of the same normal, male head specimen. The models have been incorporated into the 3D stereotactic atlases of the skull , brain , cranialTerminologia Anatomica [A process of anatomical model building was, generally, similar for each model, although varying in data acquisition, modeling methods, building suitable tools, and validation. This process included the following steps: in vivo scan acquisition, scan segmentation, scan parcellation into individual structures, 3D surface modeling, 3D model simplification (decimation or compressing), 3D surface editing, 3D model color-coding, 3D object naming based on natomica , and modnatomica , and a r2 pixel size. The skull was originally parcellated into 29 individual bones, reconstructed in 3D, and validated as featured in [In order to build a 3D model of the skull, a high-resolution spiral CT scan was acquired with 526 axial slices of 0.75 mm thickness, 0.5 mm increment, 512 × 512 matrix and 0.46875 × 0.46875 mmtured in . The sku3 voxel size. The brain was segmented by means of a dedicated contour editor, a 3D model constructed, and the cortex parcellated into lobes, gyri, and sulci [The brain model was constructed from a 3 T magnetization-prepared rapid gradient echo (MP-RAGE) scan with a 224 × 300 × 320 resolution and a 0.8 × 0.8 × 0.8 mmnd sulci .3 voxel size), spoiled gradient recovery, and susceptibility weighted imaging. The vessels were extracted using a dedicated vascular editor [In order to build a 3D model of the intracranial vasculature, multiple 3 and 7 T magnetic resonance angiography (MRA) scans were acquired, including 3D time of flight  extracted initially by means of the vascular editor from the MP-RAGE scan were also modeled as tubes, and further extended and fine-tuned as described in . The CN In order to browse, explore, navigate, quantify, and manipulate the skull base models, the atlas is equipped with a user-friendly browser with dedicated interaction. Browsing and exploration is facilitated by: (1) the assembling (compositing) and disassembling (decompositing) mechanism with an individual and group selection allowing the investigator to build any region of interest or (sub)system and to present a local anatomy within its global context; (2) virtual brain dissections in seven directions to see through the brain; (3) real-time structure and scan manipulation; (4) 3D labeling of surface and sectional MRI anatomy; (5) structure highlighting, (6) interaction combined with animation; and (7) quantification with a readout of distances, vessel diameters, and the stereotactic coordinates.The 3D, electronic and stereotactic atlas of the adult human skull base has been designed and constructed and is illustrated in Figs. The Human Brain, Head and Neck in 2953 Pieces atlas [http://www.thieme.com/nowinski/ and www.WieslawNowinski.com/FreeBrainAtlas.This herein skull base atlas is a part of es atlas , availabThis work is discussed in terms of the background in skull base presentation and modeling, skull base atlas advantages and limitations, and future directions.Photo Atlas of Skull Base Dissection by Wanibuchi et al. [Skull Base and Related Structures: Atlas of Clinical Anatomy by Lang [Atlas of Skull Base Surgery and Neurotology by Jackler [The existing skull base sources, including atlases, can be subdivided into four groups: textbooks, electronic media, 3D printed models and truly 3D electronic models. This work belongs to the last category. The skull base region has been extensively described in many sources, including textbooks of neuroanatomy , neurorai et al.  is a ric by Lang  is a tex Jackler  focuses VOXEL-MAN [A.D.A.M. Animated Dissection of Anatomy for Medicine [Interactive Head and Neck [Head and Neck Atlas [Visible Body [Visible Body models were created by medical visualization professionals and were not derived from multiple tomographic acquisitions with various contents. Generally, in the abovementioned atlases, functions for measuring distances between structures, reading vessel diameter, and providing stereotactic coordinates are not available.These limitations are partly overcome by electronic media. The skull base region or some part of it is available for viewing and manipulation in some electronic atlases of the head and neck. OXEL-MAN  compriseMedicine  consistsand Neck  containsck Atlas  containsble Body  compriseObviously, a direct manipulation and exploration of a full, truly 3D skull base model is advantageous over 2D images, animations, and teaching files. A 3D model can be solid or electronic. In , a 3D prTo our best knowledge, this is the first truly 3D atlas of the adult human skull base that includes a fully parcellated and automatically labelable brain, skull, cranial nerves, and intra- and extracranial vasculature. The use of the same specimen allowed us to build from multiple scans the 3D anatomical models that are spatially consistent, and this will enable extending them consistently in the future with new scan acquisitions. The models are polygonal (surface-based) with a higher spatial resolution in comparison with volumetric models. These component models were earlier validated, so the created skull base model can be considered as typical .This 3D atlas allows the investigator to display the entire skull base itself Figs. , 2 as weAt present, there are no predefined surgical approaches in the atlas nor dedicated surgical tools, although the available manipulation tools allow the investigator to assemble and disassemble any region, and cut it in four planes (and 7 directions) in axial, coronal, sagittal, and viewing direction planes. There is no embedded tool to capture videos; the atlas only allows the user to save the composed image to an external file. The atlas does not contain any variants of the skull base region. There are no cross-sections for the CT scans, because of a difficulty in a very accurate spatial registration of the CT scan with multiple MRI scans due to geometric distortions and various artifacts . Moreover, there are no diameters for foramina and cranial nerves.Future directions will focus on extending the current anatomical model, adding skull base variants, and including predefined surgical approaches. Moreover, we plan to develop a low cost, virtual reality surgical simulator with stereoscopic view and natural, hand-controlled content navigation for laparoscopic skull base surgeries.In summary, the created 3D interactive atlas of the adult human skull base is an easy and useful aid in studying, understanding, and teaching spatial relationships of the skull base anatomy. It allows the investigator to create any configuration, and explore and measure it, which enhances the understanding of the skull base region (including the cavernous sinus) just with a few mouse clicks, making it useful for medical students, residents, teachers, and clinicians. The atlas is a helpful tool to generate teaching materials, and a potential component of any skull base virtual reality surgical simulator.


The present meta-analysis aimed to evaluate the relationship between body mass index (BMI) and rheumatoid arthritis (RA). A systematic search of the Cochrane, Pubmed, and Embase databases was conducted to identify relevant studies published before September 2017 using terms related to BMI and RA. Fixed or random-effect models were used to estimate the pooled relative risk (RR) with 95 % confidence interval (CI). Subgroup analyses by sex were performed to investigate the association between BMI and RA in male and female subgroups. A total of 14 eligible studies containing 353,948 patients were included in the analysis. The pooled results suggested that the odds ratios (ORs) of RA were 1.08 (95 % CI: 1.00~1.15) for overweight, and 1.32 (95 % CI: 1.11~1.54) for obesity, respectively, suggesting that a higher BMI increases the risk of RA compared to normal weight. Further subgroup analyses showed a positive association between BMI and RA risk but only in females, with a RR of 1.11 (95 % CI: 1.00~1.22) for overweight and 1.40 (95 % CI: 1.24~1.57) for obesity. In conclusion, an increased BMI may lead to a higher risk for RA development. Furthermore, the positive association between BMI and RA risk may be stronger in female populations than in males. However, additional analyses are needed. Rheumatoid arthritis (RA), a common chronic autoimmune disease, is characterized by chronic, destructive, debilitating arthritis . The in2) or obese (BMI > 30 kg/m2) individuals in the USA is estimated to be two thirds of the adult population and one third of children  > 25 kg/mal., 2012; Versinial., 2012). Therefse BMI > 0 kg/m2 ial., 2012). In addal., 2012; Harpsøeal., 2012; Lahiri al., 2012; Ljung aal., 2012; Symmonsse BMI >  kg/m2 inal., 2012). This pse BMI >  kg/m2 inAlthough many studies have focused on determining the association of body mass index (BMI) and RA risk, conflicting results have been obtained and clarification is needed. Several previous studies have proposed a positive correlation between high BMI and increased risk of RA development, suggesting that obesity is a key modifiable risk factor for RA  case-control or cohort design; (2) the exposures of interest were BMI, obesity, or overweight; (3) the prevalence of RA was defined by physicians or the record linkage system, which was deemed as the outcome of interest; (4) the data included relative risk (RR) or odds ratios (ORs) with 95 % confidence intervals (CIs); (5) published in English language. If more than one article was published on the same or overlapping populations, the most recent one with the largest sample size or most detailed information was selected. A study was excluded in case of: (1) repeating publications, or studies with similar content; (2) review, case report, theoretical research, mechanism studies, conference report, systematic review, meta-analysis, unpublished, expert comment, theoretical analysis, or non-human studies; (3) the BMI was not grouped by normal weight, overweight, and obesity classifications from the WHO; (4) did not have sufficient published data or original data. All the studies were screened by two reviewers independently to determine whether they satisfied the inclusion and exclusion criteria and discrepancies were resolved by discussion with a third reviewer.Two investigators were responsible for data extraction, and the accuracy check was conducted by a third investigator. The BMI categories and RA risk estimates, including relative risk (RR) or odds ratios (ORs) with their 95 % CIs, were regarded as the primary variables of interest. RR or ORs from the maximally adjusted model was used to reduce the possibility of potential residual confounding. For each included study, data including the author name, publication year, study design, the number of patients, age of participants, and gender were extracted. The results were reviewed by two independent reviewers, and any disagreements were resolved by discussion with a third reviewer until a consensus was reached.2 are classified as overweight and those with a BMI ≥ 30 kg/m2 are characterized as obese. This meta-analysis was conducted to compare RA risk in obesity/overweight and normal BMI individuals. Stata Version 12.0 software  was used for all statistical analyses. Chi-squared and I2 tests were used to determine the significance of heterogeneity and identify the analysis model used for association: fixed-effect or random-effect model , indivial., 2005). A fixeNan, 1986). SubgroA total of 811 relevant studies were identified in the initial comprehensive literature search using index words. After screening the title or abstract, 758 articles were discarded as duplicates and 53 articles remained for full-text evaluation. After reviewing the complete manuscript, 39 articles were excluded due to following reasons: rest or exercise analysis (4), without clinical outcomes (12), no-qualified grouping 16), theoretical research (7). Only 14 studies . The pooled results suggested that being overweight could lead to a significant increase in RA risk compared with normal weight  with 299,513 participants were pooled to evaluate the association between being overweight and the risk of RA. A fixed-effect model was used to analyze the association based on the Chi-squared test P-value P = 0.546) and I and I2 t2 = 37.3 %, P = 0.188) did not exist, therefore, a fixed-effect model was used. As expected, no significant difference in the risk of RA was found between overweight and normal weight males , and thus a fixed-effect model was used for the analysis. The results indicated that the risk of RA was significantly higher in overweight females than in normal weight females , a random-effect model was used to analyze the association. Compared to normal weight individuals, obesity significantly increased the risk of RA  containing data on 301,139 participants reported the association between obesity and risk of RA. Based on the Chi-squared test P-value (P = 0.000) and I2 = 79.5 %, P = 0.002), therefore the association was assessed using a random-effect model. No significant difference in the risk of RA , therefore, a fixed-effect model was applied. In the pooled results, a significantly higher risk of RA was found in obese females compared to normal weight females  is a common chronic autoimmune disease and has been shown to be the consequence of a combination of genetic and environmental factors. Environmental risks including age, gender, obesity, cigarette smoking, alcohol consumption, and dietary habits, have been shown to be closely associated with the occurrence of this disorder . Further subgroup analysis showed that the OR for RA was higher in females  compared to males . Meanwhile, thirteen studies reported the association between obesity and risk of RA, and similar results were obtained. Collectively, it can be concluded that being overweight or obese can lead to a higher risk of RA development. In addition, the incidence of RA in women with high BMI is much higher than that in men. Compared with the findings of recent research, the results of this meta-analysis are similar. Qin et al. (2015) found aal., 2014). Secondal., 2014). HoweveThe major strengths of this meta-analysis were as follows: First, this meta-analysis was based on a comprehensive literature search of several databases to identify all relevant comparative studies. Therefore, a very large number of RA cases were included in this study, which improved the reliability, statistical power, and validity of our results. Second, most of the included studies were published in relatively high impact journals in recent years, which were of high-quality and contained more comprehensive content. Despite these strengths, several potential limitations also should also be noted. First, the included studies were limited to the English language and some qualified studies published in other languages were excluded, as were some unpublished data; thus, some publication bias existed. Second, the study populations of the included studies were limited to North American and European populations, therefore data for other continents are lacking. The development of RA is influenced by lifestyle, living environment, and economic development, thus, obesity or increased BMI may have different effects on the incidence rate of this disease in different places around the world. Based on the above, further investigations should be performed to evaluate the association between BMI and RA in different regions of the world, such as Africa and Asia. Third, RA is a multifactorial disease governed by complex gene-environment interactions and few researchers have reported on the influence of genetic factors. Therefore, we were unable to assess the effects of genetic factors on this disease. Fourth, further sensitivity analyses and publication bias analyses were unable to be performed in this study due to the lack of detailed information from the individual studies.This meta-analysis systematically assessed the association between body mass index (BMI) and rheumatoid arthritis (RA) risk based on the relevant current literature. The results demonstrated that increased BMI (classified as overweight or obese) is associated with a higher risk of developing RA. Furthermore, the positive association between BMI and RA risk may be stronger in females than in males, emphasizing sex dependency of RA. Despite these results, future work is needed to combine more risk factors in large prospective studies to reveal the mechanisms of gene-environment interactions in the development of RA. None.We all declare that we have no conflict of interest.


Xian-Ling-Gu-Bao capsule (XLGB), a famous traditional Chinese medicine prescription, is extensively used for the treatment of osteoporosis in China. However, few studies on the holistic quality control of XLGB have been reported. In this study, a reliable method using 18 representative components in XLGB was successfully established and applied to evaluate 34 batches of XLGB samples by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The choice of quantitative markers mostly followed four principles, i.e., absorbed components in plasma, bioactive compounds with in vitro anti-osteoporosis activity, those derived from multiple individual medicinal herbs in XLGB with multiple representative structure types, and quantitative chemical markers in the Chinese Pharmacopoeia. The results showed chemical consistency was good except for individual batches. Multivariate statistical analysis indicated that asperosaponin VI from Radix Dipsaci, epimedin C, magnoflorine, and icariin from Herba Epimedii as well as timosaponin BII from Rhizoma Anemarrhenae varied significantly in multiple samples, which hinted an assay for these four components should be completed during all of the manufacturing processes. Taken together, this study provided a feasible method for holistic quality control of XLGB by multiple chemical markers, which could play a vital role in guaranteeing the safety, effectiveness, and controllability of administering the capsules as a medication in clinics. Over the last few decades, traditional Chinese medicines (TCMs) have gained increasing globalization and use worldwide for human health care due to their long clinical practice in treating chronic and complex diseases ,2. As TCThe Xian-Ling-Gu-Bao capsule (XLGB), the anti-osteoporosis TCMP listed in the 2017 edition of the China National Basic Drugs Catalogue ,9. It coTo date, numerous studies concentrating on quality control of the XLGB focused on only one or two individual medicinal herbs  ,17,18,19In our previous study, sixty-one compounds were isolated and identified based on nuclear magnetic resonance (NMR) data from the bioactive fractions of the XLGB . FurtherIn this study, a total of 18 chemical markers were selected according to the following four principles: being absorbed in the plasma; possessing anti-osteoporosis activity in an in vitro test; feature representative chemical structures derived from different medicinal herbs in the capsule; and commercially available with the quantitative chemical markers for each medicinal herb in the composition recorded in the Chinese Pharmacopoeia . FurtherFor quantitative chemical markers, four main principles were followed. First, they were components absorbed in vivo. Generally, the components absorbed in vivo were considered as potential directly effective materials for therapeutic effects. As previously reported, a total of 57 prototype components were detected in rat plasma after the oral administration of XLGB extracts .Second, the quantitative chemical markers should also exhibit the same or similar activity as indicative of the TCMP. On the premise that abundant amounts of chemical components in TCM and TCMP are available, and that several components which possess activity in vitro, exhibit good absorption properties in vivo, and maintain a certain concentration in target organs for a finite period of time, then further activity evaluation in vivo could be performed. On the basis of our previous study or other related studies, several markers showed obvious anti-osteoporosis bioactivity in vitro ,23. OtheThird, in consideration of the quality control of TCMPs, the quantitative chemical markers should be derived from different individual medicinal herbs in the TCMP. The XLGB includes six common TCMs. Meanwhile, there were numerous components with multiple representative structure types, such as prenylated flavonoids and alkaloids from Herba Epimedii, saponins and iridoids from Radix Dipsaci, prenylated flavonoids and coumarins from Fructus Psoraleae, saponins from Rhizoma Anemarrhenae, and diterpenoid quinone from Radix Salviae Miltiorrhizae. To better evaluate the overall quality of the XLGB, the quantitative marker of the XLGB should be derived from as many individual medicinal herbs with multiple characteristic chemical structures as possible. Another important point is that the quantitative markers of different individual medicinal herbs in the XLGB according to the Pharmacopoeia of the Peoples’ Republic of China (2015 edition)  should bThe detailed selection principles are shown in The validation of the method included selectivity, limit of detection (LOD), and limit of quantification (LOQ), linearity, precision, repeatability, recovery, and stability tests. All measurements for method validation followed the guidelines of the Pharmacopoeia of the Peoples’ Republic of China (2015 edition) .S/N) of 3 and 10 as criteria, respectively. The linear calibration curves, plotted with a series of seven concentrations of reference standard solutions, were constructed from the peak area of each bioactive component (y) versus the concentration of each analyte (x).The limits of detection (LODs) and limits of quantification (LOQs) were measured with signal to noise ratios (r2) no less than 0.9990 within the test ranges. As shown in As a result, all the calibration curves showed good linear correlation, with the correlation coefficients  were determined. The results showed that the relative standard deviations (RSDs) of the intra- and inter-day precisions were below 3.9% and 3.7%, respectively. The detailed information is displayed in To examine the accuracy of the assay, the determination of the recovery was carried out by the standard addition method at three concentration levels. Standard mixtures of three concentration levels  were added into the sample solution (No. 110319), with three replicates for each level. Recoveries were calculated by the following formula: recovery (%) = /spiked amount × 100. Variations were expressed as the percentage RSD. The mean recoveries varied from 96.3% to 104.5% with RSDs within 4.9% shown in .In addition, the same XLGB sample (No. 110319) was analyzed at 0, 12, 24, 36, 48, and 72 h after extraction to examine the stability of the sample. The stability test showed that the sample solution was stable for 72 h with an RSD less than 3.0% .This newly developed and validated method was applied to 34 batches of XLGB samples and the quantification results are summarized in Furthermore, assay results showed that the contents of some components in each sample ranged significantly. Obviously, the total contents of several XLGB samples were higher than others, such as batch number 91104, 100523, 100832, 100840, 100846, and 100929, while the total contents of some XLGB samples  were significantly lower. The marked different consistency of multiple XLGB samples exhibited in a random pattern. As clearly shown in Chemometric analysis, using techniques such as principal component analysis (PCA), has become an effective approach in the quality evaluation of TCMs and TCMPs ,33. FromIn this study, PCA was performed with data preprocessing of scaling by the software EZinfo software of the Masslynx V4.1 workstation  based on the contents of 18 chemical markers in 34 batches of XLGB samples. A two-component PCA model was obtained which cumulatively accounted for 69.0% of the variation; the total variance explained for the first principal component is 43.9% and that for the second principal component is 25.1%. Through a visual analysis of Furthermore, the loadings plot b could cIn addition, only icariin from Herba Epimedii in XLGB samples has been considered so far as the quantitative marker for the quality evaluation in the standards by State Drug Administration. Actually, if there were only one compound considered for quality control, the holistic nature of TCMs or TCMP would not be emphasized ,35. By rThis study illustrates that quality control of XLGB is urgently needed of multiple components quantification. This would benefit the promotion of the standards by State Drug Administration. Theoretically, if at least one component of each TCM in ae XLGB capsule could be monitored and quantified in the manner accomplished in this study, then the TCM manufacturers would have to ensure the quality of every TCM, and such a loophole would be significantly eliminated, and patient care improved. Thirty-four batches of XLGB samples  that were manufactured by Guizhou Tongjitang Pharmaceutical Co., Ltd.  were collected from drugstores in Guangzhou of China. Epimedin A, epimedin B, asperosaponin VI, isobavachin, neobavaisoflavone, icariside II, bavachinin, isobavachalcone, sweroside, and corylifol A were purchased from Shanghai Winherb Medical Technology Co., Ltd. . Psoralen, timosaponin BII, isopsoralen, epimedin C, icariin, psoralidin, and tanshinone II A were obtained from Guangzhou PI&PI Inc. . Magnoflorine was obtained from Sigma-Aldrich . Their purities were all determined to be over 98% by HPLC. LC-MS grade methanol, acetonitrile and water were all purchased from Fisher Scientific . LC-MS grade formic acid was purchased from Sigma-Aldrich . Other reagents were of analytical grade.An adequate amount of each reference standard was dissolved in 60% methanol to prepare the standard solution. Subsequently, these reference standard solutions were mixed together to achieve a reasonable concentration. Before use, all of the reference standard solutions were stored at 4 °C. g for 1 min, the supernatants were filtered through a 0.22 µm membrane filter. Then, 2 µL samples were injected into the UPLC/Q-TOF-MS system. All of the solutions were stored at 4 °C before analysis. The powder of the XLGB was extracted by ultrasonic extraction with 60% aqueous methanol, and then, a final concentration of 2.5 mg/mL was achieved. After centrifugation at 13,800× TM UPLC I-Class system  equipped with a binary solvent system, an automatic sample manager, and a photo-diode array (PDA) detector. The separation was carried out on an ACQUITY UPLCTM BEH C18 column  at a temperature of 35 °C. The mobile phase consisted of mobile phase A  and mobile phase B  at a flow of 0.6 mL/min. The non-linear gradient elution program was as follows: 2% B from 0 to 0.11 min, 2–15% B from 0.11 to 4.2 min, 15–20% B from 4.2 to 4.5 min, 20–25% B from 4.5 to 9 min, 25–35% B from 9 to 15 min, 35–75% B from 15 to 16 min, 70–100% B from 16 to 16.5 min, and 100% B from 16.5 to 17.5 min. The injection volume was 2 µL. The analysis was performed on an ACQUITYTM G2 HDMS, Waters, Manchester, UK) equipped with electrospray ionization (ESI). The ionization was acquired in the positive mode. The operating parameters were as follows: capillary voltage of 3 kV (ESI+ ); sample cone voltage of 35 V; extraction cone voltage of 4 V, source temperature of 100 °C, desolvation temperature of 300 °C, cone gas flow of 50 L/h; and desolation gas flow of 800 L/h. The retention time, precursor ion, daughter ion, and collision energy under selective ion monitoring (SIM) and multiple reaction monitoring (MRM) modes are all shown in TM 4.1 software with a QuanlynxTM program. The UPLC system was coupled to a hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer (SYNAPTIn this study, a strategy of choosing quantitative markers regarding the holistic quality control of a TCMP has been successfully developed and exemplified for the simultaneous quantification of 18 representative components derived from most of the medicinal herbs of the XLGB by UPLC-Q-TOF/MS. Meanwhile, the combination of major representative chemical components from multiple individual medicinal herbs and in vivo absorbed bioactive components will be inevitably increasingly popular for the selection of quantitative markers when we hope to establish an overall quality control-oriented assay for their contents in TCMPs. In addition, this method could distinguish the consistency of multiple XLGB samples based on the quantified measurement of 18 analytes, thereby ensuring the quality of XLGB samples. Asperosaponin VI from Radix Dipsaci, icariin, epimedin C and magnoflorine from Herba Epimedii, timosaponin BII from Rhizoma Anemarrhenae, were all recommended as quantitative markers for quality evaluation of XLGB samples. These findings demonstrated that it was imperative to apply the simultaneous quantification of multiple components to the quality control of XLGB samples, so that the corresponding standards could be promoted and the quality could be ensured. This study is expected to be universally applicable to the holistic quality control of other TCMs and TCMPs.


EPM2A gene, encoding the dual phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Laforin and malin form a functional complex that is involved in the regulation of glycogen synthesis. Thus, in the absence of a functional complex glycogen accumulates in LBs. In addition, it has been suggested that the laforin-malin complex participates in alternative physiological pathways, such as intracellular protein degradation, oxidative stress, and the endoplasmic reticulum unfolded protein response. In this work we review the possible cellular functions of laforin and malin with a special focus on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.Lafora disease  is a rare and fatal form of progressive myoclonus epilepsy (PME). Among PMEs, LD is unique because of the rapid neurological deterioration of the patients and the appearance in brain and peripheral tissues of insoluble glycogen-like (polyglucosan) inclusions, named Lafora bodies (LBs). LD is caused by mutations in the  Lafora disease  is a rare and fatal form of progressive myoclonus epilepsy (PME). The term PME comprises a group of neurological disorders characterized by the presence of focal and generalized seizures, myoclonus and progressive neurodegeneration, accompanied with cerebellar signs and dementia . Among PIn this work, we review the possible cellular functions of laforin and malin, the two main proteins defective in LD, with a special focus, since malin is an E3-ubiquitin ligase, on their role in the ubiquitination of specific substrates. We also discuss here the pathological consequences of defects in laforin or malin functions, as well as the therapeutic strategies that are being explored for LD.EPM2A, encoding laforin, a glucan phosphatase, and EPM2B/NHLRC1, encoding malin, an E3-ubiquitin ligase [EPM2A gene, whereas 40–50% carry mutations in the EPM2B gene [http://projects.tcag.ca/lafora. Until June 2018, this database identified 105 modifications in the EPM2A gene  and 85 modifications in the EPM2B gene . In both genes, modifications are scattered throughout the complete sequence.LD is a very rare disease with a prevalence of less than 4 patients per 1,000,000 individuals . It is an ligase ,9,10. AcM2B gene ,7. In boEPM2A mutations are more prevalent, in others, like Italy, mutations in the EPM2B gene are more common [EPM2A, one of the most frequent mutations is R241X, whereas in the case of EPM2B, P69A is the most frequent mutation described in the literature [The geographic distribution of the mutations varies depending on the country. While in some countries, like Spain, e common . In the terature ,12.EPM2A or in EPM2B only partially affects the function of the corresponding protein, or that the presence of genetic or environmental modifiers influences the regular progression of the disease [Patients with mutations in laforin or malin are neurologically and histologically indistinguishable , suggest disease ,14,15,16EPM2A gene, containing four exons and located on the chromosome 6q24 of the human genome, which produces a major mRNA with an ORF of 993 nts, according to the ENSEMBL database. It is expressed in all tissues, but particularly in brain, skeletal muscle, heart, and liver [Laforin is encoded by the nd liver . Laforinnd liver , the CBMnd liver , being tnd liver ,21,22. Ind liver ,23,24.Laforin contains also a C-terminal DSP A. This dThe laforin crystal structure was recently described  Figure B. It conEPM2B gene, located on chromosome 6q22.3 of the human genome, which produces a major mRNA with an ORF of 1185 nts, according to the ENSEMBL database. It is expressed in brain, heart, skeletal muscle, liver, and pancreas [Malin is encoded by the single exon pancreas . Malin, pancreas   and in vitro assays have demonstrated that malin is able to interact with UBE2D1, UBE2D2, UBE2D3, and UBE2E1, but not with UBE2C and UBE2L3 [As an E3-ubiquitin ligase, malin is able to ubiquitinate specific substrates. To perform this function, malin interacts with specific E2-conjugating enzymes. Forty-one different E2-conjugating enzymes have been described thus far in the human genome  thd UBE2L3  and R6  Ad UBE2L3 ; (iv) thd UBE2L3 ; (v) pyrd UBE2L3 ; (vi) std UBE2L3 .Branchiostoma floridae [A phylogenetic study suggested that malin gene is exclusively found in vertebrate genomes and in the cephalochordate floridae . This difloridae . These rfloridae .The same report indicates that malin shares an evolutionary relationship with TRIM32, an E3-ubiquitin ligase that belongs to the tripartite motif-containing (TRIM) protein family . Both maWhen overexpressed, malin is prone to aggregate. The co-chaperone CHIP (C-terminus of Hsc70 Interacting Protein) and the chaperone Hsp70 stabilize malin . In factPatients with mutations in laforin or malin are neurologically and histologically indistinguishable . TherefoIn this complex laforin could, for example, recognize and recruit putative substrates to be ubiquitinated by malin for degradation . In factOverexpression studies with tagged version of laforin suggested that laforin has a cytoplasmic distribution, mostly associated with endoplasmic reticulum markers ,50. HoweAs mentioned above, the hallmark of LD is the accumulation in brain and peripheral tissues of poorly-branched glycogen-like inclusions named LBs. These structures also contain higher levels of phosphomonoesters on the glucose residues ,52. BothAlthough the results described above have been confirmed by different labs, they are largely based on results obtained under conditions of overexpression of laforin and malin in cell cultures using recombinant proteins. In contrast, the levels of some glycogen related proteins, such as glycogen synthase or R5/PTG, do not increase in animals of 3–6 months of age, as it would be expected by the lack of a functional laforin-malin complex ,52,67. H+ and glutamate uptake [In any case, the animal models of LD clearly show an accumulation of insoluble glycogen-like structures (polyglucosans) in different tissues . These Le uptake ,69,70. Ie uptake .Glucose enters the cell through specific glucose transporters. In the case of brain, the main glucose transporters are GLUT1 and GLUT3, expressed predominantly in astrocytes and neurons, respectively. The amount of these transporters at the plasma membrane, which is regulated by cycles of endocytosis and exocytosis, determines the efficiency of glucose uptake. It has been described  that oveIn addition to carbohydrate moieties, LBs contain up to 6% of proteinaceous material, including glycogen synthase, laforin, chaperones, autophagic components, proteasome subunits, advanced glycation end products, etc. ,62,72. MMoreover, due to the autophagy impairment, processes that require a functional autophagy machinery are also affected in cellular and animal models of the disease. In this sense, we have recently described a defect in mitophagy, most likely due again to defects in autophagosome formation  Table 2Table 2. Finally, we have also reported that oxidative stress is affected in the absence of a functional laforin-malin complex , since wWe and others have described that in animal models of LD there is a transformation of astrocytes into GFAP+ reactive astrocytes ,56. In aThe presence of reactive astrocytes and microglia is associated with pro-inflammatory conditions. In agreement with this, we have described that in the brain of animal models of LD there is an increase in the levels of pro-inflammatory mediators such as TNFα, IL-6 and IL-1β, chemokines, cytokines, and other inflammatory markers . The estWhat triggers the inflammatory process? The existence of an interrelationship between epilepsy and inflammation has been widely documented ,87,88. OIt has been reported that malin interacts with dishevelled2, a key component of the Wnt signalling pathway. Dishevelled2 is a cytosolic phosphoprotein whose activation results in the inactivation of the degradation pathway of β-catenin, resulting in the accumulation of this protein and in its translocation to the nucleus, where it activates the transcription of a specific set of genes. Overexpression of malin enhances degradation of dishevelled2 and inhibits Wnt signalling through the downregulation of β-catenin protein levels and, thus, of the expression of β-catenin target genes. Since malin enhances both K48- and K63-linked ubiquitination of dishevelled2, its degradation could occur through both proteasomes and autophagy  Table 2Table 2. Processing bodies (PBs) are cytoplasmic structures that house the RNA decay machinery . It has been reported that malin, when overexpressed, is recruited to the PBs, where it interacts with Dcp1a and promotes its degradation via the ubiquitin proteasome system . Since dThere is currently no proven therapy for LD. The initial symptoms of seizures and epilepsy are treated with regular anti-epileptic drugs , but patients soon become resistant to them . Based oIf the hallmark of the disease is the accumulation of insoluble poorly branched glycogen-like structures (LBs), it seems logical to think that any action aimed to decrease glycogen synthesis could be beneficial. This assumption was elegantly demonstrated by different authors, which created mice models combining the absence of laforin or malin and of R5/PTG or glycogen synthase. Obviously, these mice synthesized no or only small amounts of glycogen, because of the lack of key regulators of glycogen synthesis, but in addition and more interestingly, these double knock-out mice showed no symptoms of LD ,89,90,91Since proteostasis is affected in LD, other groups have analyzed the possibility of using different drugs to improve proteostasis. In this sense, since autophagy is impaired in LD, we and others checked whether inducers of autophagy, such as inhibitors of the autophagy regulator mTORC1, could be beneficial for treating this disease. However, the use of rapamycin, everolimus or temsirolimus had no effect ( and unpuFinally, additional compounds for the treatment of LD have been tested by other authors. One example is provided by sodium selenate , a compoIt has been recently described that the use of perampanel, an antagonist of post-synaptic AMPA receptors, has beneficial effects for LD in terms of decreasing the frequency of seizures and improving neurocognitive processes ,100. TheLD is a devastating pathology that leads to the death of the patients after around ten years from the beginning of the first symptoms. In recent years, many data have accumulated trying to understand the molecular basis of the pathophysiology of LD. Although several relevant discoveries have been made, the primary defect that triggers LD is still controversial. For example, we still do not understand why LD cells accumulate more glycogen than normal cells and why this polysaccharide is poorly branched and hyperphosphorylated. We also do not know how proteostasis is affected, how neuroinflammation is initiated, or how epilepsy is triggered. We hope that in the near future, we could provide answers to these yet unsolved questions. In the meantime, different strategies are being explored to obtain a treatment that slows down the progression of the disease and diminishes its symptoms.


The efficiency of metallurgical processes for separating most of the impurity elements was demonstrated, and to promote the recycling efficiency, a comprehensive management and recycling system considering the metallurgical criteria of EoL silicon wafers refining is critical.The collected end-of-life (EoL) silicon wafers from the discharged photovoltaic (PV) panels are easily contaminated by impurities such as doping elements and attached materials. In this study, the thermodynamic criteria for EoL silicon wafers refining using three most typical metallurgical refining processes: oxidation refining, evaporation refining, and solvent refining were systemically and quantitatively evaluated. A total of 42 elements  that are likely to be contained in the collected EoL silicon-based PV panels were considered. The principal findings are that the removal of aluminum, beryllium, boron, calcium, gadolinium, hafnium, uranium, yttrium, and zirconium into the slag, and removal of antimony, bismuth, carbon, lead, magnesium, phosphorus, silver, sodium, and zinc into vapor phase is possible. Further, solvent refining process using aluminum, copper, and zinc as the solvent metals, among the considered 14 potential ones, was found to be efficient for the EoL silicon wafers refining. Particularly, purification of the phosphorus doped   The u2 emissions reduction, natural resources conservation, reduction of landfilling, and reduction of heavy metals pollution [The coming boom of the EoL PV panels presents another environmental issue because they are classified as hazardous and toxic waste ,5. The Eollution ,5,7.p-type c-Si semiconductor with a heavy doped n+-type layer on the top side and heavy doped p+-type layer on the back side. Developing an efficient recycling system is becoming crucial to face the coming boom of the EoL PV panels ,5,8. By p-type semiconductors, such as boron, and group VA atoms as donors for n-type semiconductors, such as phosphorus, arsenic, and antimony [Refining the EoL silicon wafers becomes the key to close the recycling loop of the PV panels –15. Figuantimony ,18. The antimony –15,19.13+ HF + CH3COOH + Br2 or HF + HNO3 + H2SO4 + CH3COOH + CMP-MO-2 (surfactant) has been attempted to remove the metal impurities such as metal electrodes and n-p junction [Chemical etching using solutions such as HNOjunction ,20,21. Hjunction ,22–25, iThis study is meant to systemically examine the thermodynamic criteria of the metallurgical refining process of the EoL silicon wafers for closing the recycling loop of EoL c-Si PV panels. The elimination limitation of impurity elements by three most typical metallurgical refining processes: oxidation refining, evaporation refining, and solvent refining, was quantitatively evaluated using a developed thermodynamic method –33. A to2In previous studies, a thermodynamic method to evaluate the elimination limit of impurity elements contained in the EoL products of iron/steel, copper, zinc, lead, aluminum, magnesium, titanium, and Ni-Co-based superalloys was proposed –33. This2.1Lslag/silicon), and that between molten silicon and the gas phase (Lgas/silicon) [Lslag/silicon was defined as the ratio of the molar fractions of elements in slag over that in silicon, while Lgas/silicon was defined as the ratio of the partial pressure of elements in gas phase and that of silicon solvent. The calculation methods were shown as followings.The re-melting process of EoL silicon wafers with oxide-slag treatment was simulated. The elimination of a given impurity element, M, can be quantitatively presented by its equilibrium distribution ratio between molten silicon and the oxide slag (silicon) –33. Lsln in the pure standard state and its mole fraction in the slag phase; K3 is the equilibrium constant of where 2.1.1The temperature of the simulated remelting process was set to 1773 K as a base case. Thermodynamic data are available for the changes in the standard Gibbs free energy for evaporation and oxidation of the pure element M  (Supporting Information) . The actSince the concentration of impurity elements in silicon is extremely low in most cases, it is more accurate to use the activity coefficients of M in the dilute solution, p = 0, 1, 2, 3) are temperature-dependent interaction parameters.where The selection of the assessed interaction parameters for the liquid Si-M systems mainly refers to the review on the thermodynamics of elements in the dilute silicon melts by the authors .2.1.3n in slag was assumed to be unity as a first approximation [In practical metallurgical remelting process, the slag is always designed to control the activity coefficients of the oxide product in the slag. For this reason, the activity coefficients of oxides in slag have been extensively studied. In this study, the activity coefficient of MOximation –33. Furt2.1.42 . Lsolvent/silicon was defined as the ratio of the molar fractions of elements in the solvent melts over that in solid silicon, and calculated as followings.Solvent refining eliminates impurity elements according to their different affinities with solid silicon and molten solvent metals. The elimination of a given impurity element, M, by the solvent refining process is controlled by its equilibrium distribution ratio between solid silicon and molten solvent metals  = 1. In this boundary condition, the number of M atoms is 10 times as much as the number of silicon atoms in the gas phase and thus appears to be adequate for preferential removal of M into the gas phase by evaporation. The boundary between the molten silicon and slat phase is assumed to satisfy the condition log(Lslag/silicon) = 0. It is considered that in the industrial sense the removal of M into slag phase can be promoted by increasing the slag amount, repeating the oxidation refining process, and selecting the suitable slag composition.The distribution tendencies of the 42 impurity elements likely to be present in the EoL PV panels, among the molten silicon, slag, and gas phases during re-melting process are shown in 4 (g), and removal of carbon in form of CO was considered (Supporting Information). On the other hand, the other considered 24 impurity elements have a strong tendency to remain in the molten silicon phase. These trends indicate that the removal of these impurity elements by either oxidation or by evaporation during the re-melting process is difficult. The results are in good agreement with the industrial measurement results reported by a practical MG-Si refining plant in Norway, which shows that the impurity elements of aluminum, calcium, beryllium, and magnesium, can be removed into the slag phase, while zinc, lead, and some magnesium can be removed into the gas phase [During the recycling of EoL silicon wafers by re-melting process, aluminum, beryllium, calcium, gadolinium, hafnium, uranium, yttrium, and zirconium can be removed from molten silicon into the slag phase by oxidation. The removal of boron which has an almost equal distribution tendency into slag and the metal phases by slag treatment might be possible if the slag is suitable. That is, the slag used must be capable of sufficiently reducing the activity coefficient of the formed boron oxide. Antimony, bismuth, carbon, lead, magnesium, phosphorus, silver, sodium, and zinc can be separated into the vapor phase by evaporation. Noted that removal of phosphorus in form of Pas phase .The most important feature of 3.1.1Lslag/silicon ranging from 104 to 10−4 as shown in Lslag/silicon = 1 line) when temperature increases, which indicates that higher temperature is beneficial for boron removal using oxidation refining.As described in the The equilibrium oxygen partial pressures between pure silicon and pure silicon oxide at different temperatures are also shown in the top of 3.1.22, CaO-SiO2-CaF2, CaO-Al2O3-SiO2, CaO-Al2O3-SiO2-Na2O, MgO-SiO2, and CaO-SiO2-MgO, and their distribution between slag and molten silicon has been experimentally investigated [1.5 and TiOx are acidic oxides, their activity coefficients in slag with high concentration of CaO or MgO are also lower than unity [Selection of a suitable slag to promote impurity elimination by reducing the activity coefficient of the oxide products of the impurity elements, stigated –46. The affinity ,45,46. Oan unity –49.Lslag/silicon, of aluminum, calcium, magnesium, boron, and titanium between the various practical slag and molten silicon with the calculated ones [Lslag/silicon calculated by assuming Lslag/silicon for calcium, magnesium, boron, and titanium are all slightly higher than the calculated ones. Though the deviation exists, the distribution tendency for all the considered elements was not changed according to the variation of slag. Therefore, the effect of the slag composition on the removal tendency of the impurity elements is insufficient, and the evaluated results shown in Lslag/silicon close to the distribution boundary, like boron (log(Lslag/silicon) = 0.1), but have no significant effect on the elimination of other impurity elements that remain in the molten silicon phase as shown in The effect of the slag composition on the removal of the impurity elements into slag phase is found to be insignificant, though the ted ones –46. Some3.1.3Efficiency of the evaporation refining is determined by the difference of vapor pressures of impurity elements and that of molten silicon. It is found that the dependency of the elimination of the impurity elements by evaporation refining on temperature does not change significantly based on the systematic comparison of the vapor pressures of 23 impurity elements in the dilute concentration and that of the pure molten silicon at different temperatures (Supporting Information). Evaporation refining is possible to eliminate the impurity elements with higher vapor pressures than that of the molten silicon, such as phosphorus, zinc, lead, magnesium, and antimony. Meanwhile, a lower temperature is found to have slight advantages in the elimination of these impurity elements. For other impurity elements, however, evaporation refining process is not suitable.3.23.2.1Lsolvent/silicon).significantly different density from the solid silicon to promote the ease of the separation of the liquid solvent metal from purified silicon;extremely low solubility in the solid silicon to avoid contamination of the solvent metal;low eutectic/peritectic temperature close to the silicon side in the binary silicon systems;a flat liquidus on the Si-rich side of the binary silicon system to efficiently solidify silicon and to reduce the silicon loose into the solvent metal, which is indicated as the ratio of the difference of the melting point of silicon (1687 K) and the eutectic temperature, to the eutectic composition;a much lower production cost comparing with that of high purity silicon.A perfect metal for efficient solvent refining of EoL silicon wafers should have at least the following characteristics:14 metals with potential for use as solvent metals are compared in 3.2.2Lsolvent/silicon), is larger than 6, indicating an extremely strong distribution tendency into the solvent metal phase, the value of 6 will be set for such impurity element. The temperature of solvent refining process was set to 950 K, 1200 K, 1600 K, 800 K, 800 K, and 800 K for the processes that use aluminum, copper, iron, lead, tin, and zinc as the solvent metals according to the eutectic temperature, respectively.The purification of the silicon wafers from EoL PV panels by solvent refining using aluminum, copper, iron, lead, tin, and zinc as the solvent metals was systematically investigated. The thermodynamic evaluations indicate that solvent metals of aluminum, copper, zinc, and iron can efficiently eliminate the typical impurity elements contained in the EoL c-Si PV panels, including the typical dopants and the metal electrodes. The solvent refining process using aluminum, copper, and zinc as solvent metals can efficiently eliminate all the considered 13 impurity elements. Elimination of calcium using iron as the solvent metal, elimination of phosphorus using lead as the solvent metal, and elimination of boron and phosphorus using tin as the solvent metal was found difficult. Considering the high likelihood of boron and phosphorus contamination as the mostly used dopants in PV panels, lead and tin are not suitable as the solvent metals for the refining of the silicon wafers during the recycling of the EoL PV panels. However, lead and tin might be wonderful solvent metals for the refining of MG-Si owing to the high distribution ratios of iron, calcium, and titanium into the solvent metals.Lsolvent/silicon) = 17.4; iron solvent: log(Lsolvent/silicon) = 5.2; copper solvent: log(Lsolvent/silicon) = 3.4), and the removability of boron using aluminum, zinc, and iron as the solvent metals is higher  = 4.0; zinc or iron solvent: log(Lsolvent/silicon) = 3.6), respectively. Lead has the highest removability for boron (log(Lsolvent/silicon) = 6.1), however, cannot eliminate another typical dopant element phosphorus (log(Lsolvent/silicon) = −1.6). The evaluated distribution tendency of impurity elements between the molten solvent metal and solid silicon agree with the experimental ones, like that using aluminum as the solvent metal [Among solvent metals of aluminum, copper, zinc, and iron, the removability of the dopant elements in c-Si PV panels using aluminum, copper, and zinc as the solvent metals is generally higher than that using iron as the solvent metal. Particularly, the removability of phosphorus using zinc, iron, and copper as the solvent metals is higher  are completed. A comprehensive management and recycling system considering the metallurgical criteria of the refining process of the EoL silicon wafers to face the coming boom of the disposed EoL PV panels is crucial to promote the recycling efficiency.4.During the EoL silicon wafers remelting process with slag, removal of elements like aluminum, beryllium, calcium, gadolinium, hafnium, uranium, yttrium, and zirconium into the slag phase is possible. The removal of boron which have an almost equal distribution tendency into slag and the metal phases, by slag treatment might be possible if the suitable slag is used.Antimony, bismuth, carbon, lead, magnesium, phosphorus, silver, sodium, and zinc can be removed into the vapor phase by evaporation. On the other hand, removal of the other considered 24 impurity elements by either oxidation or by evaporation during the re-melting process is difficult.The effect of temperature on the elimination of impurity elements by either oxidation refining or evaporation refining is not remarkably large, while the removal of boron into the slag phase can be promoted at higher temperature.Lslag/silicon agree well with the experimentally observed ones using different practical slag (within ±2 orders), and the effect of the slag composition on the removal of the impurity elements into slag phase is found to be insignificant.The evaluated distribution ratios n-type PV panels using solvent metal zinc (log(Lsolvent/silicon) = 17.4 for phosphorus) and purification of the boron doped p-type PV panels using solvent metal aluminum (log(Lsolvent/silicon) = 4.0 for boron) are preferable.Among the considered 14 metals with potential for use as solvent metals, aluminum, copper, and zinc are found efficient for refining of EoL silicon wafers by solvent refining process, in which process all the typical dopants and the metal electrodes can be efficiently eliminated. Particularly, purification of the phosphorus doped A comprehensive management and recycling system considering the thermodynamic criteria of the refining process of the EoL silicon wafers, as summarized in For a total of 42 impurity elements that are likely to be present in the collected EoL c-Si PV panels, their elimination limitation from silicon by three most typical metallurgical refining processes, oxidation refining, evaporation refining, and solvent refining, was quantitatively evaluate. The main conclusions of this study are as follows.


Background: Maternal care refers to the behavior performed by the dam to nourish and protect her litter during its early development. Frequent and high-quality performance of such maternal behaviors is critical for the neurodevelopment of the pups. Maternal exposure to stress during early development can impair maternal care and amplify the deleterious effects of poor maternal caregiving and neglect. As such, a thorough understanding of the effects caused by several models of early life stress on maternal care may yield more insights into the relationship between stress and maternal behavior.Methods: A systematic review was performed to identify and address the effects of early life stress on maternal behavior. The search was conducted using three online databases: PUBMED, Embase, and Web of Science. To provide clear evidence of the impact of stress on maternal care, in every study, the stress group was always compared to a control group. Outcomes were categorized into eight different behaviors: (1) licking/grooming; (2) arched-back nursing; (3) blanket-nursing/passive nursing; (4) nest building; (5) contact with pups; (6) harmful/adverse caregiving; (7) no contact; (8) nest exits. Additionally, the methodological quality of the studies was evaluated.Results: A total of 12 different early life stress protocols were identified from the 56 studies included in this systematic review. Our data demonstrate that different stress models can promote specific maternal patterns of behavior. Regarding the maternal separation protocol, we observed an overall increase in nursing and licking/grooming behaviors, which are essential for pup development. An increase in the number of nest exits, which represents a fragmentation of maternal care, was observed in the limited bedding protocol, but the total amount of maternal care appears to remain similar between groups.Conclusions: Each stress protocol has unique characteristics that increase the difficulty of rendering comparisons of maternal behavior. The increase in maternal care observed in the maternal separation protocol may be an attempt to overcompensate for the time off-nest. Fragmented maternal care is a key component of the limited bedding protocol. Moreover, the methodological approaches to evaluate maternal behavior, such as time, duration, and behavior type should be more homogeneous across studies. Maternal caregiving plays a key role in childhood development , such as altered hypothalamic–pituitary–adrenal (HPA) axis functioning  reviewed the abstracts and full reports of original studies. Five exclusion criteria were applied, following a hierarchical order:Any discordance during this process was discussed with a senior scientist (RGO) to reach a final agreement about inclusion or exclusion of a given study. Furthermore, to search for any additional study, the reference list of all included papers was manually analyzed. For a detailed flow chart of the systematic review, see From each study, the following data was extracted by two independent authors : authors, publication year, strain, litter size control method, existence of cross fostering, stress period, stress model, stress duration, maternal behavior observation period, duration of maternal behavior observation, outcome variables, and methodological quality. The summary of the descriptive data obtained from all eligible studies is shown in The next step was to summarize the findings, regarding the effects of all early life stress protocols on maternal behavior. The studies were manually examined by two independent investigators (KCC and RO). To provide clear evidence of the stress impact on maternal behavior, the stress group was always compared to a control group (no stress exposure), as several studies had more than one stress group. The effects of early life stress on maternal behavior were classified as increased, decreased or no alteration on maternal caregiving  to provide conceptual information regarding specific types of maternal behaviors. The characteristics of each behavior are based on the dam's behavior and interaction with pups. To standardize the interpretation of each interaction, we divided the maternal behaviors into eight categories, based on the studies included in this review: (1) licking/grooming; (2) arched-back nursing; (3) blanket-nursing/passive nursing; (4) nest building; (5) contact with pups; (6) harmful/adverse caregiving; (7) no contact; (8) nest exits. A detailed description for each of these previously mentioned behaviors is provided in n = 410), a total of 697 papers were reviewed. Following application of exclusion criteria, 56 papers were included in this systematic review.The database search yielded 1,107 studies. After removing duplicate records  of them were performed with rats and 30.4% (n = 17) were performed with mice. When analyzing data from rat studies, we observed that Sprague-Dawley  was the primary strain used to evaluate maternal behavior. Wistar  and Long-Evans  were also commonly used strains. All other strains in this review represented only 11.91% (n = 6) of the studies, while one study did not report the utilized strain (2.38%). In 79.48% of the studies, researchers controlled the litter size (n = 31), which ranged from 6 to 14 pups, with an average of 9.8 pups per litter. In relation to the early life stress protocols utilized, the most common protocols were maternal separation , followed by limited bedding , early handling  and maternal maltreatment . The sum of all other protocols used accounted for only 12% (n = 4). Regarding the evaluation of maternal behavior, the maximum number of evaluation days was 21, and the minimum was 1. On average, there were 9 days of maternal care assessment. The daily observation of maternal behavior ranged from 30 to 480 min, with an average of 175 min of observation per day. The average time between each observation was 4.9 min.Regarding the included studies, 69.6% (n = 12). Other strains, such as BALB/c , NMRI , and CD1  were less represented. In 58.8% of studies, the researchers controlled the litter size (n = 10), and the average number of pups per litter was 6.8. Maternal separation  was the most used early life stress protocol, followed by limited bedding  and early handling . The sum of all other early life interventions accounted for 25% (n = 5). The average duration of maternal behavior evaluation for mice studies was 8.6 days, while daily observation ranged from 30 to 480 min (average of 116 min). The average time between each maternal observation was 2.26 min. The descriptive characteristics and summary of the included studies are presented in In terms of the studies using mice, we determined that the majority of studies used C57BL/6 : 30.77% for rats and 11.76% for mice. Moreover, only 12.82 and 5.88% in rats and mice, respectively, used blinding methods to collect maternal behavior. Only 25.64% of the studies with rats gave information about breeding procedures, compared to 64.71% in mice. The remaining items on the checklist were reported in at least 50% of the studies. Finally, the average percentage of all items for rat and mice studies was similar  , although no alteration and decrease were also observed in some studies , while few studies failed to report this behavior , and when it was reported, it was mainly related to its increase after early life stress exposure , which is considered a maternal behavior with no active interaction between the dam and the pups, but 5 (12.8%) of the eight studies that reported this behavior found a decrease, when comparing stressed to control dams , but 76.92% (30 studies) did not evaluate this behavior  were mostly described in terms of a decrease  or an increase , while the remaining studies did not report those behaviors  did not report nest building behavior, but no alteration was identified in three studies (17.6%), while two studies (11.8%) reported an increase in this behavior , but 12 studies (70.6%) did not report this behavior , or an increase , while five studies (29.4%) did not report this behavior  and the number of protocols (n = 70) is due to the utilization, by some studies, of more than one early life stress protocol. The majority of protocols utilized by the studies included in this review are maternal separation (MS) and limited bedding (LB). For this reason, we performed a secondary analysis to understand the specific impact of those protocols on maternal behavior. The use of MS and LB in rat and mouse studies accounted for 61.4% (43 protocols) of the 70 early life stress protocols included in this review. We identified 19 studies (38%) using MS and 11 (22%) using LB for rat studies, while MS was observed in 8 (40%) and LB in 5 (25%) protocols for studies using mice. The discrepancy between the number of studies included in the review (n = 19), we observed that the majority of the studies  reported an increase in licking and grooming behaviors , but the majority of the studies that did report it indicated no alteration  for MS protocols  or no alteration , we observed that most studies showed no alteration of licking/grooming behavior , while three studies reported an increase in these behaviors , and two studies (22.2%) showed no alteration in this behavior  did not report this behavior (Regarding the evidence for the impact of MS in mice studies (behavior . Regardibehavior . None ofbehavior .n = 11), we identified that most of the studies showed no alteration of licking/grooming behavior  of the studies did not report arched-back nursing behavior, while two studies (18.2%) indicated an increase and one study (9.1%) showed no alteration in this behavior , we noted that the only study that reported licking/grooming behavior did not observe differences between groups , nest building, licking, and grooming the pups. Observations often include non-maternal behaviors such as eating, drinking, self-grooming, or no contact with pups. It was proposed that each component of maternal behavior may reflect unique neural circuitry, but it would be biologically useful for the circuitries to overlap to regulate a group of behaviors that appear together Gammie, . In thisOne question that remains unanswered is whether early life stress alters the maternal care pattern or merely decreases the amount of time spent with the pups. It has already been shown that stress decreases the frequency of maternal LG and ABN in mothers who were previously identified as High-LG/ABN and then exposed to prenatal stress, suggesting that insults during the gestational period were able to completely reverse the maternal care pattern . Some studies performed only behavioral or biological measures (23.1% for rats and 23.6% for mice). Nevertheless, the vast majority of studies performed the follow-up by investigating behavioral and biological factors (61.5% for rats and 70.5% mice), which is critical to determine how maternal care during early development can influence several parameters throughout long-term development. Furthermore, based on the NIH directive concerning the use of females in biomedical studies , we determined that 46.2% of rat studies and 29.4% of mice studies performed sex difference comparison, while 38.4% of the rat studies and 64.7% of the mice studies used only male animals or did not report male x female comparison. Taking into account several pieces of evidence, which showed that dams tend to spend more time actively nourishing male pups , we suggest that, independently of the variations in MS protocols, an overall increase of the more important maternal behaviors, such as, nursing and licking/grooming is observed. This increase in maternal care may be an attempt to overcompensate for the time off-nest. Even though several studies used the LB model, it is very difficult to interpret the results in terms of maternal care, as most of the studies fail to report most maternal behaviors, due to methodological characteristics of the model itself. The only conclusive data, regarding LB, is that maternal behavior is fragmented during the protocol, but the amount of maternal care is not . IndepenNo datasets were generated or analyzed for this study.RO, KC, and ST conceived the study idea. RO and KC screened the abstracts and full texts. RO, LW-S, and RG-O performed data-analysis. RO, KC, LW-S, and TW wrote the manuscript. RO, KC, LW-S, TW, ST, FB, and RG-O critically revised the manuscript. RO, KC, LW-S, TW, ST, FB, and RG-O had access to all the data included in this systematic review 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. The reviewer HB declared a past co-authorship with one of the authors RG-O to the handling Editor.


ANOVA results show that, for the three references scheme, there was statistically significant difference between N170 latency and amplitude induced by the unfamiliar face and that induced by the scrambled face (p < 0.05). Specifically, the SPSM results show an anterior and a temporo-occipital distribution for AR and REST, whereas just anterior distribution shown for MM. However, the circumstantial evidence provided by source analysis is more consistent with SPSM of AR and REST, compared with that of MM. These results indicate that the experimental results under the AR and REST references are more objective and appropriate. Thus, it is more appropriate to use AR and REST reference scheme settings in future facial recognition experiments.The choice of the reference electrode scheme is an important step in event-related potential (ERP) analysis. In order to explore the optimal electroencephalogram reference electrode scheme for the ERP signal related to facial recognition, we investigated the influence of average reference (AR), mean mastoid reference (MM), and Reference Electrode Standardization Technique (REST) on the N170 component via statistical analysis, statistical parametric scalp mappings (SPSM) and source analysis. The statistical results showed that the choice of reference electrode scheme has little effect on N170 latency ( Electroencephalogram (EEG) with excellent temporal resolution is useful for determining the stages of vision-evoked information processing within the brain and plays an important role in illustrating the brains response to visual stimuli. Since the N170 component of the event-related potential (ERP) is very sensitive to configural information of the face, N170 has been regarded by researchers as a marker of face-sensitive processing . That isFor ERP studies, reference is an unavoidable problem. Since there is no zero or neutral point on the human body that can be used as a reference electrode, the EEG records the voltage between the two scalp electrodes , therebyIn order to reduce the effect of reference selection and provide a relatively neutral reference, at least with respect to the signal of interest, researchers have proposed different reference schemes, such as mean mastoid reference (MM), average reference (AR), vertex reference (Cz), and left mastoid reference (LM) . HoweverThe goal of this study was to objectively compare ERP N170 components under three different references in an attempt to determine the most suitable reference electrode for facial recognition experiments. We addressed this by comparing the ERP components referenced by AR, MM, and REST. Specifically, the N170 latency and amplitude were compared and analyzed via ANOVA; then, the SPSM of N170 was calculated. Additionally, in order to confirm the topological results, we calculated the activity source location of the cerebral cortex under different stimulation conditions and obtained the difference between the two stimuli. Then, this difference was projected back onto the scalp to obtain the corresponding EEG topographic map, serving as the standard to compare the performance of different reference electrodes. Finally, we compared the cortical source imaging with the SPSM of each reference condition, hoping that additional evidence for differences may be shown by the imaging results.1 . The scalp electrode sites were Fpz, AFz, Fz, FCz, Cz, CPz, Pz, POz, Oz, Iz, FP1/2, AF3/4/7/8, FC1/2/3/4, FC, FT7/8/9/10, C1/2/3/4/5/6, T7/8, CP1/2/3/4/5/6, TP7/8/9/10, P1/2/3/4/5/6/7/8/9/10, PO3/4/7/8/9/10, and O1/2/9/10. A 3D digitizer  was used to record the locations of the EEG electrodes, HPI coils, and approximately 50–100 “head points” around the scalp, relative to three anatomical fiducials (the nasion and left and right pre-auricular points). The EEG reference electrode was placed on the nose and the common ground electrode was placed at the left collar bone. Two sets of bipolar electrodes were used, with one set measuring the vertical and horizontal electro-oculograms (VEOG and HEOG) and the other set measuring the electro-cardiogram (ECG) of the left lower rib and right collarbone. Trials with blinks and eye movement were rejected on the basis of EOG. Data was acquired at an 1100 Hz sampling rate with a lowpass filter at 350 Hz and no high-pass filter.EEG signals were recorded using a 70 channel Easycap EEG cap and stimulate  close to the temporo-occipital was chosen as the main research objects. Previous studies  have shot-test of N170 amplitude between unfamiliar face and scrambled face conditions for each electrode scheme. The parameter was set as p < 0.01 in order to have statistically significant difference for the correction method for the FDR. The above processes were performed for the data collected with each reference scheme. In addition, the correlation of SPSM results under three references was calculated. Such analysis sought to investigate whether the scalp distribution would be affected by reference scheme selection.However, only considering the statistically significant differences of N170 latency and amplitude was not enough. Besides this, we also made further topological analysis of scalp potentials. First, all the ERP epochs induced by two types of stimuli were averaged, respectively based on AR, MM, and REST reference to obtain the ERP of each reference. Then, N170 components for different reference conditions were extracted. Finally, the scalp N170 topographic map was obtained. Topographic maps can show cortical activity level in units of uV. In addition, we performed the paired To further confirm the distribution of SPSM, minimum norm (MN) algorithm was used to illustrate the distribution of source activity for different stimuli. Firstly, the 3-shell sphere head model  of each participant was calculated, with a total of 15,003 sources (voxels) distributed around the cerebral cortex in the head model. In the source calculation process, the parameter of Regularize noise covariance is set to 0.02, the parameter of Signal-to-noise ratio is set to 3.00. The source location result shows a threshold set to 35%. Secondly, we reconstructed the source activities of averaged ERP induced by unfamiliar face stimulus and that induced by scrambled face stimulus. Thirdly, a 20 ms time window around N170 peaks in source space for the unfamiliar face and scrambled face were intercepted and averaged, and we calculated the activity intensity of the bilateral occipital lobe region of the brain induced by unfamiliar faces and scrambled faces. Finally, the averaged source activity difference obtained from the last step were projected back into sensor space to visualize the topographic map, which reflects the differences in underlying source activity. All the source reconstruction of individual ERP was performed by Brainstorm in a MATLAB environment.F = 6.66, p = 0.04,F = 44.52, p < 0.001,F = 3.23, p > 0.05,F = 2.582, p > 0.05,F = 11.01, p = 0.046,F = 3.28, p > 0.05,For the N170 amplitude, a repeated-measure ANOVA with two factors  was performed, significant main effects of reference  were comparatively investigated using statistics and topographical projections in order to find the most appropriate reference selection for facial recognition experiments. The results of the SPSM experiment  for AR aPrevious studies have shown that N170 is not affected by gender , spatialFrom the SPSM results, the distribution patterns of the scalp potential topography under the AR, MM and REST references are different. Under the AR reference, the brain regions with large differences in activity intensity are the frontal and occipital temporal regions. Under the MM reference, the brain area with a large difference in activity intensity is the frontal lobe. Under the REST reference, brain regions with large differences in activity intensity are located in the frontal and occipital temporal regions. This result is consistent with previous research results . In geneIn order to further determine which reference is more proper, analysis source activity was conducted. For EEG, the scalp distribution voltage pattern is not affected by reference, and the EEG inverse problem is independent of scalp reference . That isThus, for the proposed study, if the scalp potential distribution  projecteAccording to classical literature concerning the topic , the braIn this study, it was found that using AR and REST reference schemes showed the approximately true latency, amplitude, and SPSM mapping of N170. Compared to those referenced by MM, the ERP obtained with AR and REST references can achieve more objective analysis results, which may more accurately reflect the source activity within brain. Overall, the choice of reference is a very important and fundamental issue. This study illustrates that, at least for studies related to facial recognition, AR and REST references are clearly superior to MM references.Written informed consent was obtained from each participant prior to and following each phase of the experiment. Participants also gave separate written consent for their anonymized data to be freely available online. All materials and procedures were approved by the Cambridge University Psychological Ethics Committee.JZ and YW conceived the idea and designed the research. HY and HH developed the models and methods. TW performed the numerical calculations. YW and JZ interpreted the results and wrote the manuscript. JX, SM, HL, TW, and HH revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: The associations between the clinical characteristics and diffusion-weighted imaging (DWI) positivity in patients with a clinical diagnosis of transient ischemic attack (TIA) are still poorly understood. The purpose of our study was to determine the incidence of TIA related acute infarction by DWI, and to determine the underlying predictors of DWI positivity in TIA patients.Methods: Between Jan 2017 and Dec 2018, we retrospectively enrolled 430 patients with a clinical diagnosis of TIA who underwent DWI. Patients were divided into those with acute ischemic lesions (DWI positive group) and those without (DWI negative group). The clinical characteristics, laboratory data, and imaging parameters were compared between the two groups.Results: A total of 430 time-based TIA patients  were enrolled in this study. About 126 (29.3%) of TIA patients had a DWI positive lesion in our series. Comparing TIA patients with positive DWI to those with negative DWI, acute lesions were more likely to be more male, have higher hyperlipidemia and a smoking history, more speech abnormalities and increased motor weakness; and higher systolic and diastolic blood pressure, homocysteine, fasting blood glucose, and the scores of ABCD2, ABCD3, ABCD3-I, and Dawson. Several independent predictors of DWI positivity were identified with logistic regression analysis: motor weakness , speech abnormalities , and ABCD3-I . ABCD3-I showed the greatest area under the ROC curve, with a sensitivity of 85.7% and specificity of 72.4%.Conclusion: In patients with a clinical diagnosis of TIA, 29.3% demonstrated acute DWI lesions on brain magnetic resonance imaging (MRI). They were associated with motor weakness, speech abnormalities and higher ABCD3-I score at admission. Stroke is the second leading cause of death worldwide, and the first cause of death in China. About 20% of stroke is preceded by an episode of transient ischemic attack (TIA). Patients with TIA have the highest risk of early stroke especially in the first 48 h , 2 and sIn resource-limited settings, clinicians need to rely on patient history, neurological examination, and patient-specific risk factors to stratify. Thus, it is worthwhile to develop some reliable risk scores to prevent stroke in an early stage after the onset of TIA. The ABCD2 score was first developed to provide a structured way to stratify the early risk of stroke after TIA , 11. ABCThus, the aims of the present study are to determine the incidence of TIA related acute lesions by DWI and to investigate its utility of ABCD score system and Dawson score in our population, and finally to determine underlying predictors of DWI positivity in TIA patients.Our study was a retrospective, observational study in the Department of Neurology, Beijing Chaoyang Hospital, Capital Medical University. We retrospectively enrolled consecutive 430 patients between Jan 2017 and Dec 2018 and they all underwent brain MRI. The mean time from symptom onset to MRI was 72 h. TIA was classically defined as a focal cerebral ischemic event with neurological symptoms lasting <24 h regardless of brain lesions detected by brain imaging, including computed tomography (CT), and MRI , and administered either as a single or dual treatment.Mann-Whitney U-test. We performed a chi-square test to determine the correlation between categorical variables and a t-test between continuous variables. The multivariate logistic regression analysis was performed to find predictors for the presence of DWI lesions. The receiver operating characteristic curve (ROC) analysis was performed to discriminate DWI-positive from DWI-negative patients using the value of area under the curve (AUC). We used the Statistical Package for Social Sciences (SPSS) version 16.0  for data analysis. A P value <0.05 was considered statistically significant.The data were described using mean and standard deviation, or median and interquartile range values for continuous variables, and absolute numbers and percentages for nominal and categorical variables, and we compared the groups using the non-parametric Among a total of 430 patients with time-defined TIA, the mean age was 61.4 ± 13.0 years, and 69.5% were male. One hundred twenty-six (29.3%) patients had high-intensity lesions on DWI. We classified the patients with positive DWI as stroke etiology according to the TOAST: 48 (38.1%) of large-artery atherosclerosis, 14 (11.1%) of cardioembolism, 40 (31.7%) of small artery occlusion, 4 (3.2%) of other determined etiology, 20 (15.9%) of undetermined etiology.N = 430), DWI positive group (N = 126), and DWI negative group (N = 304). There were significant differences in sex, systolic and diastolic blood pressure, hypercholesterolemia and smoking history, motor weakness, speech abnormalities, acute antithrombotic treatment, and the scores of ABCD2, ABCD3, ABCD3-I, and Dawson between the two groups (P < 0.05). The percentage of patients who were medicated with dual antiplatelet therapy was significantly higher in the DWI positive group than DWI negative group . There were no significant differences in the other baseline clinical characteristics and MRA abnormality between the two groups.The laboratory findings were presented in CI 1.264–18.344, P = 0.021), speech abnormalities  and ABCD3I  were independently associated with positive DWI lesions.Logistic regression analysis was shown in CI) of the scores of ABCD2, ABCD3, ABCD3-I, and Dawson for the prediction of acute ischemic events were 0.629 , 0.660 , 0.854 , 0.684 , respectively. The area under the ROC curve , and Dawson was 0.684.DWI has been a mandatory tool in the diagnosis of a TIA or acute ischemic stroke . DWI is In resource-limited settings, the clinical evaluation could be quite important to stratify different risk factors of TIA patients. In patients with TIA, symptom duration of more than 1 h, motor deficits, and aphasia were independently correlated with detecting an abnormality with DWI . SubcortOur study had several limitations. First, this study was designed as a retrospective study from a single center. A study with a larger number of patients from multiple centers in China is urgently needed to confirm our results. Second, it has been reported that patients with acute DWI lesions more often performed a cardioembolic etiology or large artery atherosclerosis cause . As for In summary, our findings demonstrated that an acute infarction was detected in 29.3% of clinical diagnosed TIA patients, and it was independently associated with motor weakness, speech abnormalities, and higher ABCD3-I score at admission. A larger number of patients from multiple centers in China are urgently needed to confirm our findings, especially higher ABCD3-I score as predictors of positive DWI.wenlihu3366@126.com.The datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JY and WH conceived and designed the experiments. JY analyzed the data and drafted the manuscript. ZJ, YS, SY, YL, LY, and WQ collected data. All authors have read and approved the final manuscript to be published.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.